JP2011508793A - Controlled modification of semiconductor nanocrystals - Google Patents

Abstract

A valence-controlled semiconductor nanocrystal can have a desired number of compounds associated therewith. The semiconductor nanocrystal can have exactly one compound attached to it, and the compound can have exactly one binding site for an affinity target. The semiconductor nanocrystal can be used to image a single copy of a cell surface protein.
[Selection figure] None

Description

(Claiming priority)
This application claims priority to US Patent Application No. 60 / 946,281 filed June 26, 2007 and US Patent Application No. 60 / 990,485 filed November 27, 2007. Each is incorporated herein by reference in its entirety.
(Research or development funded by the federal government)
The US government may have certain rights in this invention in accordance with NIH grant numbers 1U65-CA119349 and 1P20GM072029-01.

(Technical field)
The present invention relates to controlled modification of semiconductor nanocrystals.

  Semiconductor nanocrystals are photostable fluorophores with narrow emission spectra that can be tuned through visible and near infrared wavelengths, large molar extinction coefficients, and high quantum yields. These properties make semiconductor nanocrystals a powerful tool for labeling and optical detection in biological, biomedical and environmental relationships. The unusual brightness and light stability of semiconductor nanocrystals give them great potential for the analysis of biological events at the single molecule level. However, current nanocrystals designed for cell labeling applications suffer from incompatible adjustments between size, non-specific binding, and derivatization ability.

  In general, nanocrystals comprise inorganic nanoparticles surrounded by a layer of organic ligands. This organic ligand shell is indispensable for nanocrystals for processing, bonding to certain other sites, bonding to various substrates. Nanocrystals can be stored for extended periods in their growth solutions containing large excesses of ligands such as alkyl phosphines and alkyl phosphine oxides without significant degradation. For most applications, especially aqueous applications, nanocrystals must be processed outside their growth solution and transferred to various chemical environments. However, when removed from their growth solution, nanocrystals often lose their high fluorescence or are irreversibly aggregated.

(Summary)
Single particle tracking can provide an unprecedented understanding of cell surface protein behavior without simplification of the ensemble average. However, single particle tracking is complicated by fluorophore photobleaching, probe multivalency, labeled probe size, and probe dissociation from the target. Semiconductor nanocrystals have inherent advantages with respect to brightness and photostability single particle tracking. Multivalency can be avoided by purifying semiconductor nanocrystals that bind to a single copy of monovalent streptavidin. The size of the semiconductor nanocrystal can be equivalent to that of an IgG antibody. Probe dissociation can be minimized by using monovalent streptavidin to link nanocrystals to site-specific biotinylated cell surface proteins. Monovalent semiconductor nanocrystals can be used, for example, to image neurotransmitter receptors in synapses and to follow the diffusion of wild-type or disease-related mutant low density lipoprotein receptors.

  In one aspect, a method for producing a valence-controlled semiconductor nanocrystal comprises contacting a population of semiconductor nanocrystals with a compound having an affinity for the semiconductor nanocrystal to form a distribution of compound-bound nanocrystals. And separating the members of the distribution of compound-bound nanocrystals according to the number of compounds bound to each nanocrystal.

  The method can include isolating a member of the distribution of compound-bound nanocrystals in which exactly one compound is bound to the nanocrystal. The compound can selectively bind a ligand. The compound can selectively bind exactly one ligand. The compound can be avidin or streptavidin. The compound can be monovalent avidin or monovalent streptavidin. The compound can include a polyhistidine tag. The compound can be an antibody, such as a single chain antibody.

The semiconductor nanocrystal can include an outer layer comprising a compound of formula (I):
R ¹ -L ¹ -R ² -L ² -R ³
(I) in which R ¹ is optionally one or more of -O-, -S-, -C (O)-, -N (R ⁴ )-or -C (O) N (R ⁴ )- Linear or branched C ₁ -C ₁₀ alkyl, alkenyl, or substituted with two or more groups selected from hydroxy, thiol, amino, nitrogen oxide, phosphine or phosphine oxide; Alkynyl chain. L ¹ is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O) —, —C (O) N (R ⁴ ) —O— or — (CR ⁵ R ⁶ ) _n —. R ² is-[(CR ⁵ R ⁶ ) _n -X- (CR ⁵ R ⁶ ) _n ] _m- , X is O, S, C (= O) or N (R ⁴ ); and m Is an integer in the range 0-20. L ² is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O) —, —C (O) N (R ⁴ ) —O— or — (CR ⁵ R ⁶ ) _n —. R ³ is — (CR ⁵ R ⁶ ) pR ⁷ , R ⁷ is —COOH, —OP (O) (OH) OH, amino, alkylamino, dialkylamino or trialkylamino; and p is 0, 1, 2, 3, 4, 5, or 6. R ⁴ is H or C ₁ -C ₆ alkyl. Each R ⁵ and each R ⁶ is independently selected from H, hydroxy, amino, thio, nitro, alkylamino, dialkylamino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl and heteroaryl; and each n Are independently 0, 1, 2, 3, 4, 5, or 6.

  In another embodiment, the method of imaging a single particle comprises binding an affinity tag to a cell surface protein; the cell has exactly 1 binding site for the affinity tag. Contacting with a composition comprising a controlled valence semiconductor nanocrystal associated with a compound; and imaging the cell and the semiconductor nanocrystal substantially simultaneously.

  The affinity tag can be biotin. Binding the affinity tag to the cell surface protein can include contacting a fusion protein comprising a receptor peptide (AP) sequence with a biotin ligase. The semiconductor nanocrystal can bind to exactly one monovalent avidin or exactly one monovalent streptavidin.

又は下記式：

を有することができる。 The semiconductor nanocrystal includes an outer layer comprising the compound of formula (I) described above.
The compound of formula (I) has the following formula:

Or the following formula:

Can have.

In another embodiment, a semiconductor nanocrystal comprising an outer layer comprising a compound of formula (I) as described above. R ¹ can be HS—CH ₂ CH ₂ CH (SH) — (CH ₂ ) ₄ —. R ² can be poly (alkylene oxide). R ² can be poly (ethylene glycol). R ² can have the formula — [CH ₂ —O—CH ₂ ] _m —, where m is about 8. R ³ can be —CH ₂ —R ⁷ where R ⁷ is amino, alkylamino, dialkylamino or trialkylamino. R ⁷ can be —COOH. R ³ can be —CH ₂ COOH.

In some embodiments, R ¹ is _{HS-CH 2 CH 2 CH (} SH) - (CH 2) 4 - , R ² is poly (alkylene oxide), and R ⁷ is -COOH, amino, Alkylamino, dialkylamino or trialkylamino.

又は下記式：

を有することができる。 The compound has the following formula:

Or the following formula:

Can have.

  Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

FIG. 1A is a graph showing optical characteristics of semiconductor nanocrystals. FIG. 1B is a graph showing the physical properties of semiconductor nanocrystals. FIG. 2A is a graph showing the electrostatic properties of semiconductor nanocrystals. FIG. 2B is an electrophoresis gel image using semiconductor nanocrystals with different ligands. 3A-3E are fluorescent images and photographs of cells exposed to different semiconductor nanocrystals. FIG. 4A is a schematic diagram of the binding of a dye to a semiconductor nanocrystal. 4B-4C are graphs showing the optical properties of semiconductor nanocrystals. 4D to 4F are graphs showing elution profiles of standard samples and semiconductor nanocrystals during gel filtration chromatography. FIG. 5 is a schematic diagram in which cell surface proteins are labeled with semiconductor nanocrystals. 6A-6C are synthetic photographs / fluorescence images of cells. FIG. 6D is a graph showing a single molecule diffusion coefficient. FIG. 7A is an image of an electrophoresis gel. 7B-7E are synthetic photographs / fluorescence images of cells. FIG. 8A is a schematic diagram in which cell surface proteins are labeled with semiconductor nanocrystals and fluorescence detection via FRET. FIG. 8B is a fluorescence image of the cells. FIG. 9A is a schematic diagram of semiconductor nanocrystals with different affinity labels. FIG. 9B is a schematic diagram in which a single cell surface protein is labeled with a single semiconductor nanocrystal. Figures 10A-10B are images of electrophoresis gels showing the isolation of semiconductor nanocrystals bound to exactly one streptavidin. FIG. 11A is an AFM image showing a semiconductor nanocrystal. FIG. 11B is a fluorescent image and photograph of the cells. FIG. 12 is a fluorescent image and photograph of the cells. FIG. 13 is a fluorescence image of cells. 14A-14B are images of electrophoresis gels representing the isolation of semiconductor nanocrystals bound to exactly one antibody. FIG. 14C is a fluorescent image and photograph of the cells. 15A-15B are fluorescent images and photographs of the cells. FIG. 16 is a schematic diagram in which cell surface proteins are labeled with semiconductor nanocrystals. FIG. 17 is a series of fluorescent images and photographs of cells. FIG. 18 is a fluorescent image and photograph of the cells.

(Detailed explanation)
The ultimate goal of cell imaging is to observe single biomolecule changes in living cells. In vitro single molecule imaging has proven its ability to detect low levels of biomolecules such as miRNA or viral particles and become the ultimate in sensitivity (eg, Neely, LA et al., (2006) Nat. Methods 3, 41-46; and Agrawal, A. et al., (2006). Anal. Chem. 78, 1061-1070, each of which is incorporated by reference in its entirety. ). In vitro single molecule imaging methods detect temporary conformational changes during enzyme turnover, and kinesin steps below the diffraction limit of light, avoiding the averaging inherent in ensemble methods. For example, English, BP et al. (2006). Nat. Chem. Biol. 2, 87-94; Kusumi A. et al. (2005). Annu. Rev. Biophys. Biomol. Struct. 34, 351-378; And Yildiz, A., Selvin, PR (2005). Acc. Chem. Res. 38, 574-582. Each of which is incorporated by reference in its entirety. On the other hand, single molecule imaging of cells (eg Saxton, MJ, Jacobson, K. (1997). Annu. Rev. Biophys. Biomol. Struct. 26, 373-399, which is incorporated by reference in its entirety). Is a major challenge, and an unchanging battle against weak and unstable fluorescent signals from dyes or fluorescent proteins is under way (eg, Tardin, C. et al. (2003). EMBO J. 22, 4656). -4665; Lommerse, PH et al. (2004). Biophys. J. 86, 609-616; and Douglass, AD, Vale, RD Cell 121, 937-950, each of which is incorporated by reference in its entirety. .) Dye-labeled Fab molecules are monovalent, but are dim, easily fade, and exhibit weak binding. In some cases, a total reflection illumination fluorescence (TIRF) microscope is used to detect a single fluorescent protein in living mammalian cells. However, the fluorescence typically fades within 10 seconds. Gold particles or latex beads allow for stable single particle imaging, but are typically 30-500 nm in diameter. In recent years, gold size has been reduced to 5 nm by detecting local heating during gold irradiation rather than light scattering (see, for example, Lasne, D. et al. (2006). Biophys. J. 91, 4598-4604. (It is incorporated by reference in its entirety.) This is a promising development, but 5nm meant a gold nucleus, after which it was passivated and bound to primary and secondary antibodies.

  Semiconductor nanocrystals offer certain advantages in tracking single molecule changes in cells. Single semiconductor nanocrystals are extremely light-stable with sufficient brightness to be easily recognized by a wide-range fluorescence microscope without the need for TIRF (Gao, X. et al. (2005 Curr. Opin. Biotechnol. 16, 63-72, which is incorporated by reference in its entirety.). However, semiconductor nanocrystals have a size, unstable bond between the nanocrystal and the protein of interest, and nanocrystal multivalency (i.e., a large number of nanocrystals on the nanocrystal such that the nanocrystal can cross-link with multiple targets). The problem of the presence of target binding sites. For example, Groc, L. et al. (2004). Nat. Neurosci. 7, 695-696; Howarth, M. et al. (2005). Proc. Natl. Acad. Sci. USA 102, 7583-7588; See Jaiswal, JK and Simon, SM (2004) Trends Cell Biol. 14 [9], 497-504, each of which is incorporated by reference in its entirety. Conventional nanocrystals are 20-30 nm in diameter, which can reduce diffusion in crowded cell locations such as cytosol or synapses. The unstable binding of the nanocrystal to the target protein is usually due to a monovalent antibody that dissociates from its target in a few minutes. This is too short to accurately track the most dynamic processes in living cells (see Schwesinger, F. et al. (2000). See Proc. Natl. Acad. Sci. USA 97, 9972-9977. (This is incorporated by reference throughout.)

  The multivalency of nanocrystals (as well as other nanoparticles such as gold nanoparticles) is a significant concern: cross-linking of surface proteins activates signal pathways and promotes contact with the cytoskeleton As a result, the receptor mobility can be reduced. See, for example, Klemm, J.D. et al. (1998). Annu. Rev. Immunol. 16, 569-592; and Iino, R. et al. (2001). Biophys. J. 80, 2667-2677. They are incorporated by reference in their entirety. The excessive addition of nanocrystals does not completely solve the cross-linking problem. This is because the target protein can dissociate from one nanocrystal and crosslink to another nanocrystal. Also, for example, the pool of target proteins in the recycling endosome cannot access the initial pulse of the nanocrystal, reach the surface on another nanocrystal and crosslink to its free binding site. obtain. Complete dissolution for possible cross-linking requires nanoparticles with uniformly single binding sites.

One method for preparing monovalent nanoparticles is based on electrophoretic separation by mobility shift from different numbers of DNA ligands. For example, Fu, A. et al. (2004). J. Am. Chem. Soc. 126, 10832-10833; Qin, WJ, Yung, LY (2005). Langmuir 21, 11330-11334; and Ackerson, CJ et al. (2005). See Proc.Natl.Acad.Sci.USA 102, 13383-13385. Each of which is incorporated by reference in its entirety. Methods for controlling protein valency based on DNA will require subsequent DNA removal if the nanoparticles are not very large. Another method is based on low density functional sites on polystyrene beads, where one ligand on the particle can be activated for ligand binding (see, eg, Sung, KM et al. ( 2004) J. Am. Chem. Soc. 126, 5064-5065; and Worden, JG et al. (2004). Chem. Commun. (Camb.) 518-519, each of which is cited in its entirety. Is taken in.) This method only gave monovalence to ˜60%. The purification of gold nanoparticles according to the number of His ₆ tagged peptides using a nickel affinity column is shown. However, there was a significant overlap in the elution profiles of monovalent and multivalent particles (Levy, R. et al. (2006). Chembiochem. 7, 592-594, which is incorporated by reference in its entirety). Another method is to add a ligand such as biotinylated epidermal growth factor (EGF) to the streptavidin-nanocrystal in a substoichiometric ratio. Most nanocrystals do not bind EGF, some bind one EGF, and rarely bind two EGF molecules (Lidke, DS et al. (2005). J. Cell Biol. 170, 619-626, which is incorporated by reference in its entirety.) This is an inefficient use of nanocrystals and if the biotinylated ligand contains more than one biotin group, the nanocrystals can crosslink with each other.

  Optimal design of semiconductor nanocrystals for single-molecule imaging of living cells is an inherent challenge. Nanoparticles feature easy derivatization through multiple linking strategies, low non-specific binding, low hydrodynamic size, high quantum yield, and the ability to form agglomeration-free water dispersion over a wide pH range Would be.

  Currently, the dominant class of nanocrystals used for single molecule cell imaging are those that retain a hydrophobic surface ligand from synthesis and are encapsulated within an amphiphilic polymer shell. For example, Wu, X. et al. Nature Biotechnol. 2003, 21, 41-46; Medintz, I. et al. Nature Mater. 2005, 4, 435-446; Zhou, M. et al. Bioconjugate Chem. 2007, 18, 323-332; Dahan, M. et al. 2003, 302, 442-445; and Courty, S. et al. Nano. Lett. 2006, 6, 1491-1495. Each of which is incorporated by reference in its entirety. Such encapsulated nanocrystals benefit from high quantum yield (QY). However, polymer shells form large aggregates of about 20-30 nm due to the inorganic core / shell diameter of only 4-6 nm (see, for example, Smith, AM et al. Phys. Chem. Chem. Phys. 2006, 8, 3895 -3903, which is incorporated by reference in its entirety.) The size of polymer-coated nanocrystals was often much larger than labeled targets, which was a major obstacle to widespread use of nanocrystals in biological imaging. This potentially interferes with the function of the labeled protein and restricts access to disordered spaces such as neuronal synapses. See, for example, Howarth, M. et al., PNAS. 2005, 102, 7583-7588; and Groc, L. et al., Nat Neurosci 2004, 7, 695-696. Each of which is incorporated by reference in its entirety. Furthermore, in many cases, the amphiphilic polymer coating is highly filled. This causes non-specific binding to the cell membrane, making it unsuitable for single particle imaging where low background is essential. Non-specific adsorption can be mitigated through PEGylation of polymer-encapsulated nanocrystals, which further increases nanoparticle size (see, for example, Bentzen, EL et al. Bioconjugate Chem. 2005, 16, (See 1488-1494, which is incorporated by reference in its entirety.) Nanocrystals coated with phospholipids or silica shells are also used in biological systems, but suffer from similar limitations on the inherent large size and the need for a bulky PEG passivation layer. See, for example, Dubertret, B. et al., Science 2002, 298, 1759-1762; and Parak, W. et al., 2002, 14, 2113-2119. Each of which is incorporated by reference in its entirety.

  Replacing the native hydrophobic coating with a small molecule ligand containing a carboxylic ester such as mercaptoacetic acid (MAA) can greatly reduce the size of the nanocrystals while maintaining derivatization capability. . For example, Aldana, J. et al., J. Am. Chem. Soc. 2001, 123, 8844-8850; Mattoussi, H. et al., J. Am. Chem. Soc. 2000, 122, 12142-12150; See S. paper Bawendi, MG, J. Am. Chem. Soc. 2003, 125, 14652-14653; and Algar, WR paper Krull, UJ, 2006, 22, 11346-11352. Each of which is incorporated by reference in its entirety. Although such nanocrystals have a hydrodynamic diameter (HD) of only ~ 6-8 nm, they can be inherently unstable due to weak ligand-nanocrystal interactions, Nanocrystal precipitation occurs on a time scale of several hours under ambient conditions. See, for example, Smith, AM et al., Phys. Chem. Chem. Phys. 2006, 8, 3895-3903; and Aldana, J. et al., J. Am. Chem. Soc. 2001, 123, 8844-8850. I want. Each of which is incorporated by reference in its entirety. In addition, the ionization of the carboxylate group necessary to make the nanocrystals water-dispersible results in instability under acidic conditions and promotes nonspecific binding to cells (Bentzen, EL et al. Bioconjugate Chem. 2005, 16, 1488-1494; and Xue, F. et al. Journal of Fluorescence 2007, 17, 149-154, each of which is incorporated by reference in its entirety. Furthermore, nanocrystals that are ligand-exchanged with such monothiol-based ligands suffer a significant decrease in quantum yield (Smith, AM et al., Phys. Chem. Chem. Chem. Phys. 2006, 8, 3895). -3903, which is incorporated by reference in its entirety.) Dithiol ligands such as dihydrolipoic acid (DHLA) are more stable to ligand dissociation, but still form nanocrystals that precipitate under mildly acidic conditions. See, for example, Mattoussi, H. et al., J. Am. Chem. Soc. 2000, 122, 12142-12150; and Pons, T. et al., J. Phys. Chem. B 2006, 110, 20308-20316. I want. Each of which is incorporated by reference in its entirety. Furthermore, DHLA coated nanocrystals show high non-specific binding and cannot be used in single particle tracking applications. Ligand exchange with esters of DHLA with various lengths of PEG formed nanocrystals that were very stable in aqueous solution and suitable for live cell imaging (e.g. Uyeda, HT et al. J Am. Chem. Soc. 2005, 127, 3870-3878, which is incorporated by reference in its entirety. However, the hydroxyl-terminated surface of these DHLA-PEG nanocrystals under mild conditions, for example, for efficient and selective covalent derivatization with targeted biomolecules for receptor labeling on cells Lacks sensuality.

Two commonly used nanocrystal derivatization strategies are 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mediated crosslinking between amino and carboxyl functional groups Direct covalent modification of nanocrystals using common bioconjugation methods such as coupling, and self-assembly of biomolecules via electrostatic or metal affinity interactions on the nanocrystal. See, for example, Wu, X. et al. Nature Biotechnol. 2003, 21, 41-46; Yan Zhang et al. Angew. Chem. Int. Ed. 2006, 45, 4936-4940; and Goldman, ER et al. See Am. Chem. Soc. 2002, 124, 6378-6382. Each of which is incorporated by reference in its entirety. Nanocrystals encapsulated in a polymer / phospholipid / silica shell are usually derivatized by covalent linkage. Nanocrystals capped with DHLA or DHLA-PEG are susceptible to linkage using metal-affinity interactions between His _6- tagged biomolecules and the metal surface of the nanocrystals, and nanocrystal emission of coordinated biomolecules And results in stable conjugates that retain functionality (see, eg, Medintz, IL, Nature Mater. 2003, 2, 630-638, which is incorporated by reference in its entirety). Currently, there is a lack of a powerful strategy that combines the covalent and His ₆ -tag binding capabilities on a single nanoparticle while maintaining the properties of small size, low non-specific binding, and solution stability.

  The nanocrystal ligand can include a first portion that includes one or more coordination atoms, a second portion that includes one or more hydrophilic groups, and a third portion that includes one or more ionic groups. . The coordinating atom can be, for example, N, O, P or S. A coordinating atom can be included in a coordinating group (eg, amine, nitrogen oxide, alcohol, carboxylate, thiocarboxylate, phosphine, phosphine oxide, thiol, etc.). Hydrophilic groups can include, for example, alkoxides, amines, thiols, alcohols, carboxylates, ketones, aldehydes, and the like. An ionic group can be a group having electrostatic charge or a group that can be electrostatically charged in an aqueous environment. Typical ionic groups include amines (eg, primary, secondary, tertiary, and quaternary amines), carboxylates, alcohols, thiols, and the like.

  In some embodiments, the first, second and third portions of the ligand are each derived from a different precursor compound. The first portion can include two or more coordination atoms, such as 2, 3, 4, 5, 6 or more coordination atoms. In some embodiments, the first portion has two coordinating atoms. The second portion can be substantially derived from poly (ethylene glycol) or poly (propylene glycol).

R ¹ -L ¹ -R ² -L ² -R ³ (I)
Wherein R ¹ is optionally interrupted by one or more of -O-, -S-, -C (O)-, -N (R ⁴ )-or -C (O) N (R ⁴ )-. And a straight or branched C ₁ -C ₁₀ alkyl, alkenyl, or alkynyl chain substituted by two or more groups selected from hydroxy, thiol, amino, nitrogen oxide, phosphine or phosphine oxide is there.

L ¹ is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O) —, —C (O) N (R ⁴ ) —O— or — (CR ⁵ R ⁶ ) _n —.
R ² is-[(CR ⁵ R ⁶ ) _n -X- (CR ⁵ R ⁶ ) _n ] _m- , X is O, S, C (= O) or N (R ⁴ ); and m Is an integer in the range 0-20.

L ² is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O) —, —C (O) N (R ⁴ ) —O— or — (CR ⁵ R ⁶ ) _n —.
R ³ is — (CR ⁵ R ⁶ ) _p —R ⁷ , R ⁷ is —COOH, —OP (O) (OH) OH, amino, alkylamino, dialkylamino or trialkylamino; and p Is 0, 1, 2, 3, 4, 5, or 6.
R ⁴ is H or C ₁ -C ₆ alkyl.

Each R ⁵ and each R ⁶ is independently selected from H, hydroxy, amino, thio, nitro, alkylamino, dialkylamino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl and heteroaryl. Each n is independently 0, 1, 2, 3, 4, 5, or 6.

式中、nは範囲1〜100、1〜50又は1〜20である。nの値は約8である。すなわち、配位子の個々の分子は整数値のみを有することができるが、配位子のバルク試料においては、nは整数値の分布を有することができ、nの平均値が約8である。 In some embodiments, the ligand can have the following formula:

Where n is in the range 1-100, 1-50 or 1-20. The value of n is about 8. That is, the individual molecules of the ligand can only have integer values, but in the bulk sample of the ligand, n can have an integer value distribution and the average value of n is about 8. .

An efficient means for coating nanocrystals with amine or carboxy functional group terminated DHLA-PEG is described. These nanocrystals have excellent photophysical properties and a size substantially smaller than encapsulated nanocrystals. These nanocrystals can be linked via covalent bond formation and His ₆ -protein coupling for target cell labeling applications. The quantum yield (QY) of these DHLA-PEG derivatized nanocrystals was enhanced through the alloy ZnCd1-xSx shell, giving a QY as high as 45% after ligand exchange. These nanocrystals exhibit very low non-specific binding to cells and have excellent pH stability. The nanocrystals were also used for live cell imaging by targeting the low density lipoprotein receptor, a protein whose transport has an important role in preventing coronary artery disease. In addition, a biotin ligase for nanocrystal targeting that uses a monovalent variant of streptavidin as a high affinity and low valent bridge to stably link nanocrystals to specifically biotinylated receptors (See, for example, Howarth, M. et al. PNAS. 2005, 102, 7583-7588; and Howarth, M. et al. Nat Meth 2006, 3, 267-273, each of which Incorporated by reference throughout.)

  Nanocrystal cores can be prepared by pyrolyzing organometallic precursors in a high temperature coordinating agent. See, for example, Murray, C.B. et al., J. Am. Chem. Soc. 1993, 115, 8706, and Mikulec, F., Ph.D. Thesis, MIT, Cambridge, 1999. Each of which is incorporated by reference in its entirety. Growth of the shell layer on the bare nanocrystal core can be performed by a simple modification of the conventional overcoating procedure. For example, Peng, X. et al., J. Am. Chem. Soc. 1997, 119, 7019, Dabbousi, BO et al., J. Phys. Chem. B 1997, 101, 9463, and Cao, YW and Banin, U. Angew. Chem. Int. Edit. 1999, 38, 3692. Each of which is incorporated by reference in its entirety.

  Coordinating agents can help control the growth of nanocrystals. A coordinating agent is a compound having a donor lone electron pair, for example, having a lone electron pair that can be used to coordinate to the surface of a growing nanocrystal. The coordinating agent can be a solvent. Solvent coordination can stabilize the growing nanocrystals. Exemplary coordinating solvents include alkyl phosphines, alkyl phosphine oxides, alkyl phosphonic acids, or alkyl phosphinic acids, although other coordinating solvents such as pyridine, furan, and amines may also be suitable for nanocrystal formation. . Examples of suitable coordinating solvents are pyridine, tri-n-octylphosphine (TOP), tri-n-octylphosphine oxide (TOPO), and tris-hydroxylpropylphosphine (tHPP). Industrial TOPO can be used.

  The outer surface of the nanocrystal can include a layer of a compound derived from the coordinating agent used during the growth process. The surface can be modified by repeated exposure to excess competing coordination groups to form a coating layer. For example, the dispersion of nanocrystals capped with the coordinating agent used during growth is treated with a coordinating organic compound such as pyridine and is easily dispersed in pyridine, methanol, and aromatics, but aliphatic. Crystals that are no longer dispersed in the solvent can be produced. Such a surface exchange process can be performed with any compound that can coordinate or bind to the external surface of the nanocrystal, including, for example, phosphines, thiols, amines, and phosphates. Nanocrystals can be exposed to short chain polymers that terminate at sites that have an affinity for the surface and an affinity for the suspension or dispersion medium. Such affinity improves the stability of the suspension and prevents nanocrystal aggregation.

  Monodentate alkyl phosphines and alkyl phosphine oxides efficiently passivate nanocrystals. Note that the term phosphine relates to phosphine and the following phosphine oxides. Other conventional ligands such as thiols or phosphonic acids may be less effective than monodentate phosphines that maintain the initial high nanocrystal emission over time. For example, the photoluminescence of nanocrystals is consistently attenuated or quenched after ligand exchange with thiol or phosphonic acid.

  Ligand exchange can be carried out by one-phase or two-phase methods. Prior to ligand exchange, nanocrystals can be precipitated from their growth solution by the addition of methanol. Supernatant containing excess coordinating agent (eg, trioctylphosphine) can be discarded. The precipitated nanocrystals can be redispersed in hexane. The precipitation and redispersion can be repeated until essentially all the excess coordinating agent has been separated from the nanocrystals. If the nanocrystal and the introduced ligand are soluble in the same solvent, a single phase process can be used. Solutions with excess new ligand can be mixed with the nanocrystals. The mixture can be stirred at an elevated temperature until ligand exchange is complete. One-phase methods can be used, for example, to exchange octyl-modified oligomeric phosphine or methacrylate-modified oligomeric phosphine, both of which are dissolved in a solvent miscible with nanocrystals, such as hexane. If the nanocrystal and the new ligand do not have a common solvent, a two-phase ligand exchange process is preferred. Nanocrystals can be dissolved in an organic solvent such as dichloromethane and new ligands can be dissolved in an aqueous solution. Nanocrystals can be transferred from the organic phase to the aqueous phase, for example, by sonication. The movement can be monitored by absorption and emission light analysis. Similar biphasic ligand exchange processes have been reported previously. See, for example, Wang, Y.A. et al. 2002 J. Am. Chem. Soc 124, 2293, which is incorporated by reference in its entirety.

  The nanocrystal may be a member of a population of nanocrystals having a narrow size distribution. Nanocrystals can be spherical, rod-shaped, disc-shaped, or other shapes. The nanocrystal can include a core of semiconductor material. The nanocrystal can include a core having the formula MX, where M is cadmium, zinc, magnesium, mercury, aluminum, gallium, indium, thallium, or mixtures thereof, where X is oxygen, sulfur, Selenium, tellurium, nitrogen, phosphorus, arsenic, antimony, or mixtures thereof.

  Semiconductors that form the core of the nanocrystal are II-VI compounds, II-V compounds, III-VI compounds, III-V compounds, IV-VI compounds, I-III-VI compounds. Group II, IV-VI group, or II-IV-V group compounds such as ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgO, MgS, MgSe, MgTe, HgO, Contains HgS, HgSe, HgTe, AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, TlN, TlP, TlAs, TlSb, PbS, PbSe, PbTe, or mixtures thereof be able to.

  The core can have an overcoating on the surface of the core. The overcoating may be a semiconductor material having a composition different from the composition of the core. The overcoat of semiconductor material on the surface of the nanocrystal is made up of II-VI compounds, II-V compounds, III-VI compounds, III-V compounds, IV-VI compounds, I- III-VI compounds, II-IV-VI compounds, or II-IV-V compounds such as ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgO, MgS, MgSe, MgTe , HgO, HgS, HgSe, HgTe, AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, TlN, TlP, TlAs, TlSb, PbS, PbSe, PbTe, or those Mixtures can be included. The overcoating material can have a band gap that is larger than the band gap of the core material. Alternatively, the overcoating material can have an intermediate band in energy (ie, valence band or conduction band) in the valence band and conduction band of the core material. See, for example, US Patent Application Publication No. 20040110002, entitled “Semiconductor Nanocrystal Heterostructures,” which is incorporated by reference in its entirety.

  The emission from the nanocrystal can be a narrow Gaussian emission band, and by changing the size of the nanocrystal, the composition of the nanocrystal, or both, the ultraviolet, visible, or infrared It can be tuned over the entire wavelength range of the spectrum of the region. For example, CdSe can be adjusted in the visible region and InAs can be adjusted in the infrared region.

  The population of nanocrystals can have a narrow size distribution. The population can be monodisperse and can exhibit a deviation of less than 15% rms, preferably less than 10%, more preferably less than 5% of the nanocrystal diameter. For nanocrystals that emit light in the visible, a narrow range of spectral emission can be observed with a full width at half maximum (FWHM) in the range of about 10 nm to 100 nm. Semiconductor nanocrystals can have emission quantum efficiencies greater than 2%, 5%, 10%, 20%, 40%, 60%, 70% or 80%.

  A method for preparing semiconductor nanocrystals involves pyrolysis of an organometallic reagent such as dimethylcadmium injected into a hot coordinating solvent. This allows for individual nucleation and results in controlled growth of macroscopic nanocrystals. The preparation and manipulation of nanocrystals is described, for example, in US Pat. No. 6,322,901, the entirety of which is incorporated herein by reference. The manufacturing method of nanocrystals is a colloidal growth process and can produce a monodisperse particle population. Colloidal growth occurs by rapidly injecting M and X donors into a hot coordinating solvent. This implantation produces nuclei that can be grown in a controlled manner to form nanocrystals. The reaction mixture can be gently heated to grow and anneal the nanocrystals. Both the average size and the size distribution of the nanocrystals in the sample depend on the growth temperature. The growth temperature required to maintain stable growth increases with increasing average crystal size. Nanocrystals are members of a group of nanocrystals. As a result of individual nucleation and controlled growth, the resulting population of nanocrystals has a narrow monodisperse diameter distribution. The monodisperse diameter distribution can also be called size. The process of controlled growth and annealing of nanocrystals in a coordinating solvent following nucleation can also result in uniform surface derivatization and regular core structure. As the size distribution becomes sharper, the temperature can be raised to maintain stable growth. By adding more M donor or X donor, the growth period can be shortened.

  The overcoating process is described, for example, in US Pat. No. 6,322,901. The entirety of which is incorporated herein by reference. By adjusting the temperature of the reaction mixture during overcoating and monitoring the absorption spectrum of the core, an overcoated material with high emission quantum efficiency and narrow size distribution can be obtained.

The M donor can be an inorganic compound, an organometallic compound, or an elemental metal. The inorganic compound M-containing salt can be a metal diketonate such as a metal halide, metal carboxylate, metal carbonate, metal hydroxide or metal acetylacetonate. See, for example, US Pat. No. 6,576,291. It is incorporated by reference throughout. M is cadmium, zinc, magnesium, mercury, aluminum, gallium, indium, or thallium. An X donor is a compound that can react with an M donor to produce a substance having the general formula MX. Typically, the X donor is a chalcogenide donor or pnictide donor, such as a phosphine chalcogenide, bis (silyl) chalcogenide, dioxygen, ammonium salt, or tris (silyl) pnictide. Suitable X donors are dioxygen, bis (trimethylsilyl) selenide ((TMS) ₂ Se), trialkylphosphine selenides (eg (tri-n-octylphosphine) selenide (TOPSe) or (tri-n-butylphosphine)) Selenide (TBPSe), etc., trialkylphosphine telluride (eg (tri-n-octylphosphine) telluride (TOPTe) or hexapropylphosphorustriamide telluride (HPPTTe), etc.), bis (trimethylsilyl) telluride ((TMS) ₂ Te), bis (trimethylsilyl) sulfide ((TMS) ₂ S)), trialkylphosphine sulfide (eg (tri-n-octylphosphine) sulfide (TOPS) etc.), ammonium salt (eg ammonium halide (eg NH ₄ Cl ), Tris (trimethylsilyl) phosphide ((TMS) ₃ P), tris (trimethyl) Silyl) arsenide ((TMS) ₃ As) or tris (trimethylsilyl) antimonide ((TMS) ₃ Sb). In certain embodiments, the M donor and the X donor can be components within the same molecule.

  The size distribution during the growth phase of the reaction can be evaluated by monitoring the absorption line width of the particles. Modification of the reaction temperature in response to changes in the absorption spectrum of the particles makes it possible to maintain a sharp particle size distribution during growth. In order to grow larger crystals, reactants can be added to the nucleation solution during crystal growth. Growth can be stopped at a specific nanocrystal average diameter, resulting in a population having an average nanocrystal diameter of less than 150 mm. The population of nanocrystals has an average diameter ranging from 15 to 125 mm.

  As described in US Pat. No. 6,322,901, which is incorporated by reference herein in its entirety, the particle size distribution is determined by size selective precipitation using a poor solvent for nanocrystals such as methanol / butanol. Can be further purified. For example, nanocrystals can be dispersed in 10% butanol in hexane. Methanol can be added dropwise to the stirred solution until the opalescence persists. Separation of the supernatant and aggregates by centrifugation produces a precipitate enriched in the largest crystals in the sample. This procedure can be repeated until no further sharpening of the absorption spectrum is observed. The size selective precipitation method can be carried out with various solvent / non-solvent pairs including pyridine / hexane and chloroform / methanol. The size-selected nanocrystal population can have a deviation of 15% rms or less from the average diameter, preferably 10% rms or less, more preferably 5% rms or less.

  Transmission electron microscopy (TEM) can provide information about the size, shape, and distribution of nanocrystal populations. Powder X-ray diffraction (XRD) patterns can provide the most complete information regarding the type and quality of the crystal structure of nanocrystals. Further, since the particle diameter is inversely proportional to the peak width via the X-ray coherence length, the size can be estimated. For example, the diameter of the nanocrystal can be measured directly by transmission electron microscopy or can be estimated from X-ray diffraction data using, for example, Scherrer's equation. It is also possible to estimate from the UV / Vis absorption spectrum.

  A valence-controlled semiconductor nanocrystal can have a desired valence for a binding site for an affinity target. Therefore, semiconductor nanocrystals are exactly 1, exactly 2, exactly 3, exactly 4, exactly 5, exactly 6, exactly 7, exactly 8, It can be prepared to have exactly 10, exactly 11, exactly 12, or exactly a larger number of binding sites. A population of semiconductor nanocrystals can be prepared in which each of the semiconductor nanocrystals in the population has exactly the desired valence. Monovalent semiconductor nanocrystals can have exactly one binding site for an affinity target.

  Valence-controlled semiconductor nanocrystals can be prepared by linking compounds to semiconductor nanocrystals. In some embodiments, the linking chain produces a probability distribution of the number of compounds that bind to individual semiconductor nanocrystals. The distribution can be influenced by changing the relative amount of compound and semiconductor nanocrystals present at the time of linking. Distributions with different numbers of compounds binding to semiconductor nanocrystals (e.g. 0 compounds, exactly 1 compound, exactly 2 compounds, exactly 3 compounds or exactly a higher number of compounds) Members can be separated from each other. The separation can be a size-based separation (eg, electrophoresis or gel filtration chromatography) or an affinity-based separation (eg, affinity chromatography). Affinity chromatography can include generating a column having a fixed ligand (ie, an affinity target) to which the compound binds. The affinity target can be linked to a chromatographic resin having a reactive functional group according to methods known in the art. Nanocrystals bound to the compound can be immobilized with the resin by virtue of the interaction between the compound and the affinity target. Nanocrystals that do not bind to the column are not fixed and can be efficiently removed by washing the resin. The resin is then washed by elution conditions (e.g., changes in pH, ionic strength, temperature, or exposure to competing ligands or ligand analogs) that weaken the interaction between the compound and the nanocrystal. The Furthermore, increased exposure to elution conditions weakens the interaction so that nanocrystals that are bound to exactly one compound are eluted from the resin before nanocrystals that are bound to exactly more than one compound.

This interaction may be a specific binding interaction such as maltose that binds an antibody that binds its target antigen or a protein that binds to amylase; an electrostatic interaction such as a highly charged peptide that binds to a cation or anion exchange column; or It may also be a metal-ligand interaction such as a nickel or cobalt column that binds to a polyimidazole tag (eg, His ₆ tag).

  In some embodiments, a population of semiconductor nanocrystals can be prepared in which substantially all of the semiconductor nanocrystals are bound to exactly one compound. In some embodiments, the compound has exactly 1 binding site, exactly 2 binding sites, exactly 3 binding sites or exactly 4 binding sites for the affinity target. . In this way, a population of semiconductor nanocrystals can be prepared in which each portion of the population of semiconductor nanocrystals can bind to an exact number of binding sites ranging from 0 to 12 or more for affinity targets.

  The compound can be, for example, avidin or streptavidin. The avidin or streptavidin can have one or more monomers having a modified amino acid sequence such that the one or more monomers are substantially unable to bind biotin. The avidin or streptavidin can be monovalent avidin (mA) or monovalent streptavidin (mSA). The compound can be an antibody, single chain antibody, or other protein or peptide sequence having specific binding affinity for an affinity target, e.g., avimer or affibody (e.g. Biz HK et al., Nat Biotechnol. 2005 Oct; 23 (10): 1257-68, which is incorporated by reference in its entirety. An affinity target can be, for example, a protein, nucleic acid, peptide, metabolite, or small molecule.

  Cell surface proteins can be labeled with nanocrystals by antibody targeting (FIG. 9A). Methods for site-specific biotinylation of cell surface proteins using biotin ligase are known (eg, Howarth, M. et al. (2005). Proc. Natl. Acad. Sci. USA 102, 7583-7588; and Chen, I. et al. (2005). See Nat. Methods 2, 99-104, each of which is incorporated by reference in its entirety.) Biotin ligase rapidly biotinylates the lysine side chain in the 15-amino acid receptor peptide (AP) sequence genetically added to the protein of interest. See, for example, Beckett, D. et al., Protein Sci. 1999 Apr; 8 (4): 921-9. It is incorporated by reference throughout. The biotinylated AP-tagged protein can then be stably tracked at the single molecule level using streptavidin-coated nanocrystals (Howarth, M. et al. 2005). The high stability of streptavidin labeling avoids the minute order of reversibility that is characteristic of most antibody-antigen interactions (Green, NM (1990). Methods Enzymol. 184, 51-67. Incorporated by reference). However, the size and multivalency of the nanocrystals used in previous studies are obstacles to obtaining reliable quantitative data on receptor diffusion and transport (e.g., Groc, L., (2004)). Nat. Neurosci. 7, 695-696; and Howarth, M. et al. (2005). See Proc. Natl. Acad. Sci. USA 102, 7583-7588, each of which is incorporated by reference in its entirety. ing.). Commercially available streptavidin-nanocrystals have 4-10 copies of tetravalent streptavidin per particle (see, eg, Grecco, HE et al. (2004). Microsc. Res. Tech. 65, 169-179. (It is incorporated by reference in its entirety.) This gives 16-40 possible biotin binding sites per nanocrystal. In addition, up to 30% of certain commercially available nanocrystals can be dimerized or trimerized (Nehilla, BJ et al. (2005). J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys, which is incorporated by reference in its entirety.)

  In order to avoid cross-linking when labeling cell surface proteins with tetrameric streptavidin, streptavidin with one biotin binding site rather than four was developed (e.g. Howarth, M. et al. Nat. Methods 3, 267-273; and US Patent Application Publication No. 2007/0099248, entitled “Monovalent streptavidin compositions,” each of which is incorporated by reference in its entirety. Is taken in.) This streptavidin had the same binding affinity, off-rate and heat resistance as wild-type streptavidin, but no cross-linking occurred when used for labeling cell surface proteins.

  The modified streptavidin monomer can have a modification of the core wild type streptavidin amino acid sequence. The modification of the streptavidin subunit sequence is a change in the amino acid sequence of the streptavidin monomer subunit from the wild-type amino acid sequence. Modification of a streptavidin amino acid sequence can include substituting one or more amino acid residues of that sequence with another amino acid sequence, wherein one amino acid sequence is another in the mature sequence of wild-type streptavidin. (Residues 25-163 of SEQ ID NO: 1) is an example of modification of the streptavidin subunit. Residue 25 of the sequence shown as Genbank accession number P22629 (SEQ ID NO: 1) is considered to be one residue of the mature wild-type streptavidin monomer, and residue 37 of SEQ ID NO: 1 is the wild-type core It is thought to be a single residue of the streptavidin sequence. Using this numbering method, the residues that are altered in the preparation of some modified monomers of the present invention include residues N23, S27 and S45. An example of a modified streptavidin monomer subunit is the D subunit, which includes the following substitutions: N-> A at position 23 of the amino acid sequence of the mature wild-type streptavidin monomer; amino acid sequence of the mature wild-type streptavidin monomer S-> D at position 27 of S; and S-> A at position 27 of the amino acid sequence of the mature wild-type streptavidin monomer.

Wild-type streptavidin:

The sequence described as a dead (D) monomer sequence includes the following substituted amino acid residues: N23A, S27D and S45A (numbering based on the numbering of the mature wild-type streptavidin sequence, which is the amino acid of SEQ ID NO: 1 1 to 163). The dead monomer sequence is described herein as SEQ ID NO: 3. The sequence of the above alive (A) monomer subunit has an unmodified core wild type streptavidin monomer sequence and may also include a His ₆ purification tag. In some embodiments, the alive (A) monomer does not have a purification tag. The alive streptavidin monomer subunit with a His ₆ tag is described herein as SEQ ID NO: 4. For use in some methods and preparations of the invention, the sequences of SEQ ID NOs: 2, 3, and 4 are encoded in the plasmid with the starting methionine and then removed by E. coli. It will be appreciated that the presence of the initiating methionine is not a modification of the core sequence and is therefore not a modification.

Dead Monomer:

Alive monomer:

Alive monomer with His ₆ tag:

  Monovalent streptavidin (mSA) tetramer was modified from one wild type streptavidin monomer subunit or fragment thereof that maintains wild type streptavidin binding affinity to biotin, and the wild type streptavidin amino acid sequence Contains three modified streptavidin subunits having amino acid sequences. One type of modified streptavidin subunit used in the monovalent streptavidin tetramer of the present invention is referred to herein as the dead (D) type streptavidin monomer subunit and is referred to as the modified streptavidin subunit. It is a monomer subunit. Preferred monovalent streptavidin tetramers of the present invention comprise wild type and modified streptavidin subunits in a ratio of 1: 3. Monovalent streptavidin tetramer contains only a monofunctional biotin-binding subunit. In some preferred monovalent streptavidin tetramers, all three modified streptavidin monomer subunits have the same type of sequence modification.

  The wild type streptavidin monomer subunit can also include a purification tag that can be used for the preparation and purification of monovalent streptavidin polymers. The wild type streptavidin monomer subunit can include a purification tag that is not required but can be used for the preparation and purification of monovalent streptavidin polymers consisting of: One example of an alternative method for purifying monovalent streptavidin polymers without using a purification tag is the separation of various tetramers using an iminobiotin column. In the iminobiotin column, the D4 tetramer does not bind to the column, the other streptavidin tetramers elute in the order of A1D3, A2D2, A3D1, then the pH is lowered, and A4 with the later tetramer elutes To do. One skilled in the art will recognize that additional methods can be used for the separation and / or purification of streptavidin tetramers having different ratios of A and D streptavidin subunits.

Nanocrystals coated with DHLA-PEG-CO ₂ H have a negative surface charge, migrate faster in agarose gel electrophoresis, and have a mobility similar to 1 kilobase DNA (FIG. 2B). His _6- tagged proteins can be efficiently linked to these nanocrystals by multidentate interaction between the imidazole group and the Cd / ZnS shell of the nanocrystal (Clapp, AR et al. (2004 J. Am. Chem. Soc. 126, 301-310, which is incorporated by reference in its entirety.). When DHLA-PEG-CO ₂ H nanocrystals were incubated with different concentrations of monovalent streptavidin containing a single His ₆ -tag and analyzed by electrophoresis, a ladder of significant nanocrystal mobility was obtained. The observed discontinuous and sharp bands correspond to different numbers of streptavidin linked to the nanocrystals (FIG. 10A). Such a ladder was previously observed after the protein was linked to the nanocrystal, but no purification was shown. For example, Pinaud, F. et al. (2004). J. Am. Chem. Soc. 126, 6115-6123; and Pons, T. et al. (2006). J. Phys. Chem. B Condens. Matter Mater. See Surf. Interfaces. Biophys. 110, 20308-20316. Each of which is incorporated by reference in its entirety. Electrophoretic separation allowed a simple calibration of the optimal streptavidin concentration to produce nanocrystals with a single monovalent streptavidin (mSA) (ie monovalent nanocrystal) per nanocrystal. The ligation of mSA was almost quantitative, unlike the yield <10% usually obtained for EDC ligation of His-tagged proteins to these nanocrystals. Furthermore, monovalent nanocrystals could be easily purified by excising visible bands from the gel and separating the nanocrystals from agarose by centrifugation. After recovery of these monovalent nanocrystals, no nanocrystals bound to 0 or 2 copies of streptavidin were detected when retested on an agarose gel (FIG. 10B). This indicated that the monovalent nanocrystal was isolated with good purity.

(Materials and analysis)
All chemicals were obtained from Sigma Aldrich and used as received. Air sensitive materials were handled in an Omni-Lab VAC glove box under a dry nitrogen atmosphere with <0.2 ppm oxygen level. All solvents were spectrophotometric grade and purchased from EMD Biosciences. Compounds containing amines were visualized on thin layer chromatography (TLC) plates using ninhydrin solution. All other TLC plates were visualized by iodine staining. NMR spectra were recorded on a Bruker DRX 401 NMR spectrometer. ESI-MS was measured with an Applied Biosystems QTrap mass spectrometer (Forster City, CA). The sample was dissolved in a solution of acetonitrile, water and acetic acid (50: 50: 0.01 v / v) at a concentration of 2.5 pmol / μL and introduced via a syringe pump at a flow rate of 20 μL / min. All poly (ethylene) glycol compounds produced molecular weight distribution and separated at 44 m / z. The maximum mass of the distribution is reported in the ESI-MS characterization of all samples. UV-Vis absorbance spectra were obtained using an HP 8453 diode array spectrophotometer. Photoluminescence spectra were recorded with a SPEX FluoroMax-3 spectrofluorometer. The absorbance of all solutions was kept below 0.1 OD to avoid internal filter effects. Zeta potential measurement was performed with a Zeta PALS instrument (Brookhaven Instruments).

(Gel filtration device)
GFC was performed using an Amersham Biosciences AKTAprime Plus chromatography system equipped with a Superose 6 10 / 300GL column. Phosphate buffered saline (1X PBS) was used as the mobile phase at a flow rate of 0.5 mL / min. The typical injection volume was 50 μL. Detection is achieved by measuring absorption at 280 nm, and fluorescence spectra are simultaneously measured at set time intervals using an Ocean Optics SD2000 fiber optic spectrometer with excitation at 460 nm from an Ocean Optics LS-450 LED light source. Recorded. The column was calibrated in the range of MW 1.3 to 158 kDa using gel filtration protein standards (cat. 151-1901) from Bio-Rad.

(Dynamic light scattering)
Light scattering analysis was performed using DynaPro Dynamic Light Scatterer. All nanocrystal samples were at a concentration of 0.5-2 μM and were filtered through a 0.02 μm filter prior to analysis. A typical count rate was 85-150 kHz. Each autocorrelation function (ACF) was obtained for 10 seconds and averaged for 10 minutes per measurement. A software filter was used and all ACF fits were truncated with a sum of squared errors> 15. The resulting ACF was fitted using Dynamics V6 software using a non-negative least squares fitting algorithm. Hydrodynamic size data was obtained from mass weighted size distribution analysis and reported as the average of triplicate measurements.

(Solubilization of CdSe (ZnCdS) core (shell) nanocrystals in water)
The exchange of natural TOPO / TOP surface ligands on nanocrystals with PEG derivatized ligands was performed according to previously reported procedures in several variations (Uyeda, HT et al. J. Am. Chem. Soc. 2005, 127, 3870-3878, which is incorporated by reference in its entirety. Acetone was added to 0.2 mL of the nanocrystal growth solution until it became cloudy. After centrifugation and decanting, 50 μL of neat DHLA-PEG derivatized ligand and 10 μL of MeOH were added. The mixture was stirred at 60 ° C. for 2.5 hours and precipitated by sequential addition of 0.3 mL ethanol, 0.05 mL chloroform, and 0.5 mL hexane. The supernatant was obtained by centrifugation at 3000 g for 2 minutes and discarded. The pellet was dispersed in 0.5 mL PBS and filtered through a 0.2 μm filter. The sample was clarified using gel filtration chromatography to remove aggregated nanocrystals and the fractions were concentrated at 3500 g using a Vivaspin-6 10,000 spin concentrator. The typical concentration of nanocrystal preparation was 8 μM. The water QY was ˜40%.

(Quantum yield measurement)
The QY of the 565 nm emitting nanocrystal was measured against rhodamine 590 with excitation at 490 nm. Nanocrystals in PBS and dye in ethanol were optically matched at the excitation wavelength. QD and dye fluorescence spectra were obtained in triplicate and averaged under the same spectroscopic conditions. Emission QY _NC = (absorbance) _dye / (absorbance) _NC x (peak area) _NC / (peak area, using the integrated intensity of the emission spectrum calibrated for differences in refractive index and concentration, keeping the optical density of the peak below 0.1 ) _Dye x (n _{NC solvent} ) ² / (n _{Dye solvent} ) ² x QY _dye was used to calculate the quantum yield (see, eg, Eaton, D., IUPAC. 1988, 60, 1107-1114. Incorporated by reference throughout.)

(Diamino-PEG (Compound 1))
The synthesis of the ligand is also illustrated in Scheme 1 below. Neat poly (ethylene glycol) (average MW 400) (20.0 g, 48.3 mmol) was degassed with stirring at 80 ° C. for 1 hour to remove traces of water. The flask was filled with N _2, cooled in an ice bath, thionyl chloride (10.51mL, 145.0mmol) was added slowly. The solution was warmed to room temperature and stirred for 2 hours. The conversion, disappearance of broad OH stretch of 3500 cm ^-1 in the IR spectrum, and was monitored by the appearance of C-Cl stretch of 730 cm ^-1. The product was diluted with DMF (20 mL) and the solvent was removed under reduced pressure. This was repeated 3 times to remove any residual thionyl chloride. The sample was dissolved in 250 mL of DMF solution of sodium azide (9.42 g, 145.0 mmol) and stirred at 85 ° C. overnight. The solvent was removed under reduced pressure and 200 mL of dichloromethane was added. The precipitate was removed by vacuum filtration and the solvent was removed under reduced pressure to give the intermediate azide functionalized product. The conversion was confirmed by the appearance of a sharp azide stretch of 2100 cm ^{−1 in} the IR spectrum and the disappearance of the C—Cl stretch of 730 cm ⁻¹ . The sample was dissolved in 300 mL of tetrahydrofuran and triphenylphosphine (27.9 g, 106 mmol) was added. The solution was stirred at room temperature for 4 hours, 4 mL of water was added and stirred overnight. The solvent was removed under reduced pressure and 100 mL of deionized water was added. The precipitate was removed by vacuum filtration and the filtrate was washed with toluene (3 × 50 mL). The solvent was removed under reduced pressure to give the pure product as a pale yellow oil (15.5 g, 78%).

(NHC ester of thioctic acid (TA-NHS))
To a 150 mL THF solution of lipoic acid (5.00 g, 24.23 mmol) and NHS (3.35 g, 29.1 mmol), 10 mL of a THF solution of DCC (6.00 g, 29.1 mmol) was added at 0 ° C. The mixture was warmed to room temperature and stirred for 5 hours. The precipitate was removed by vacuum filtration, and the solvent was distilled off under reduced pressure. The crude product was redissolved in 100 mL ethyl acetate and filtered again by vacuum filtration. The product was recrystallized from a hot ethyl acetate: hexane (1: 1 v / v) solution as a pale yellow solid (5.88 g, 80%).

(TA-PEG-NH ₂ (Compound 2))
Lipoic acid-NHS (1.60 g, 5.27 mmol) at 0 ° C. in a DMF / water (100 mL, 50:50 v / v) solution of compound 1 (12 g, 29.1 mmol) and sodium bicarbonate (2.44 g, 29.1 mmol). ) Was added dropwise over 1 hour. The solution was warmed to room temperature, stirred overnight and extracted with chloroform (3 × 30 mL). The combined organic extracts were washed with water (3 × 30 mL), dried over Na ₂ SO ₄ , filtered and evaporated. The crude product was purified by alumina column (dichloromethane / methanol 95: 5) to give the final product as a yellow oil (1.90 g, 60%).

(TA-PEG-CO ₂ H (Compound 5))
To a dichloromethane solution (30 mL) of compound 2 (1.90 g, 3.16 mmol) and triethylamine (0.320 g, 3.16 mmol), a dichloromethane solution (10 mL) of methylmalonyl chloride (0.475 g, 3.48 mmol) was slowly added dropwise at 0 ° C. . The solution was stirred at room temperature for 4 hours and the solvent was removed under reduced pressure. The crude product was purified by silica column (dichloromethane / methanol 95: 5) and evaporated to give the pure product (compound 3) as a yellow oil (1.97 g, 89%). Deprotection of the methyl ester was achieved by stirring at 60 ° C. for 5 hours with 3.5 equivalents of NaOH in methanol. The solvent was neutralized to pH 7 with 3M HCl and then removed under reduced pressure. The product was dissolved in water, acidified to pH 2, and extracted with chloroform (3 × 20 mL) to give the pure product in quantitative yield.

(General procedure for disulfide ring opening (compounds 3 and 6))
To a 0.25M NaHCO ₃ solution (20 mL) of TA-PEG, 4 equivalents of sodium borohydride were slowly added over 30 minutes at 0 ° C. The solution was stirred on ice for 2 hours, acidified to pH 2 with 3M HCl and extracted with chloroform (3 × 15 mL). The combined organics were dried over MgSO ₄ and filtered. The solvent was removed under reduced pressure to give the product as a colorless oil.

(Agarose gel electrophoresis)
Electrophoresis of nanocrystals was performed using a Minicell Primo (Thermo) with 1% Omnipur agarose (EMD) in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.3) for 15 minutes at 7.9 V / cm. It was. Nanocrystals were diluted to 150 nM with TAE, mixed with 6x loading buffer (16% sucrose) and loaded onto the gel. The gel was visualized under 305 nm UV using a ChemiImager 5500 (Alpha Innotech). Monovalent streptavidin in PBS was prepared as previously described (Howarth et al. 2006). Wild type streptavidin was dissolved at 1 mg / mL in PBS. To test the effects of nanocrystal-linked His ₆ -tag, 5 μL of 3 μM nanocrystals in sodium borate pH 7.4, emitting at 600 nm and coated with 10 mM DHLA-PEG-CO ₂ H, in PBS Incubated with 5 μL of 27 uM monovalent streptavidin for 1 hour at 24 ° C. Subsequently, streptavidin ligation was examined by electrophoresis. These nanocrystals were used at 50 nM for cell labeling.

(Purification of monovalent nanocrystals)
0.5 μL of 8 μM nanocrystals in PBS were incubated with indicated amounts of monovalent streptavidin (19 μM) or scFv (13 μM) in PBS for 1 hour at 24 ° C. in a total volume of 5 μL. Monovalent streptavidin was prepared as disclosed (Howarth et al. 2006) mSA and scFv each contained a single His ₆ -tag that stably binds to the Cd / ZnS shell (Clapp et al. (Reference, 2004) Addition of 1-ethyl-3-diisopropylaminocarbodiimide (EDC) did not change the coupling efficiency. Analysis of nanocrystal-protein linkage was performed using a Minicell Primo (Thermo) with 1% Omnipur agarose (EMD) in 10 mM sodium borate (adjusted to pH 8.0 with 1 M HCl) for 15 min at 7.9 V / Performed by electrophoresis at cm. Samples were loaded with 6x loading buffer (16% sucrose in ddH ₂ O). For purification, the buffer was cooled on ice, the electrophoresis apparatus was enclosed in ice, and the gel was run at 6.4 V / cm for 20 minutes. Samples for purification of monovalent nanocrystals were generated using a molar ratio of 1 nanocrystal to 1 mSA. Gels were visualized using an analytical ChemiImager 5500 (Alpha Innotech) under 305 nm UV or visually under ambient light for purification. The band of interest was excised with a scalpel and placed on a Nanosep MF 0.2 μm column (Pall) and centrifuged at 5,000 g for 3 minutes at 24 ° C. This centrifugation rotated the buffer and nanocrystal-protein conjugate from agarose into the lower collection tube (see, eg, Loweth, CJ et al. (1999). Angewandte Chemie-International Edition 38, 1808-1812. (It is incorporated by reference throughout.) The extraction efficiency from agarose was 30-50% (data not shown).

(Linking monovalent streptavidin dye-functionalized nanocrystals)
To link streptavidin to the nanocrystals, 5 μL of 3 μM control or dye-linked nanocrystals in 10 mM sodium borate pH 7.4 was incubated at 24 ° C. with 5 μL of 27 uM monovalent streptavidin in PBS. These nanocrystals were then used at 50 nM for cell labeling.

及び

を用いて逆PCRによって、pEGFP-LDLRに挿入した。EGFPを、プライマー

及びその逆補体を用いてQuikChange(商標)により除去した。構築物を、配列決定によって検査した。 (Cell culture, labeling and imaging)
HeLa (human cancer) and COS7 (African green monkey kidney) cells were grown in DMEM with 10% fetal calf serum, 50 U / mL penicillin and 50 μg / mL streptomycin. Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and cells were imaged the next day after transfection. pEYFP-H2B (Platani Swedlow 2002) was a kind gift from AK Leung (MIT). This encodes histone H2B fused to EYFP as a nuclear local cotransfection marker. LDLR-AP has an AP inserted after the signal sequence of the human low density lipoprotein receptor, allowing extracellular display. LDLR of pEGFP (Clontech) was a kind gift of T. Kirchhausen (Blood Research (Center for Blood Research, Harvard Medical School). Cytoplasmic cotransfection marker BFP was a gift from RY Tsien (UCSD). The human EGFR gene in pcDNA3 (Invitrogen) was a gift from KD Wittrup (MIT).

as well as

Was inserted into pEGFP-LDLR by inverse PCR. EGFP, primer

And its reverse complement were removed by QuikChange ™. The construct was examined by sequencing.

  CHO ldlA7 (A7) is a variant of CHO that lacks endogenous LDLR and was a kind gift from M. Krieger (MIT, US). CHO, HeLa and A7 were grown in DMEM with 10% fetal calf serum, 50 U / mL penicillin, 50 μg / mL streptomycin, 1 mM pyruvate and 69 mg / L with L-proline. DMEM has been reported to contain no biotin (Invitrogen) and 100% fetal bovine serum contains ~ 90 nM biotin (see, eg, Baumgartner, MR et al. (2004). Am. J. Hum. Genet. 75, 790 See -800, which is incorporated by reference throughout.) The cell line was transfected with 1 μl of Lipofectamine 2000 (Invitrogen), 0.1 μg of the gene of interest and 5 ng cotransfection marker per well of a 48 well plate. When cells were co-transfected with BirA-ER and AP fusions, 0.1 μg of each plasmid was used per well. Cells were imaged the next day after transfection. Primary hippocampal cultures were prepared from E18-19 rats according to MIT guidelines. Briefly, hippocampus was separated with papain and neurons were plated at a density of 200,000 cells / well onto a 12 mm coverslip of Eagle basal medium (BME) plus 5% fetal calf serum. Coverslips were precoated with poly-D-lysine (Mw 300,000) (Sigma) (0.1 mg / ml) and mouse laminin (Invitrogen) (5 μg / ml). After the culture was grown for 8 hours at 37 ° C, the medium was supplemented with Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamine, 25 U / mL penicillin, and 25 μg / mL streptomycin for further culture. Was replaced. Neurons were transfected with calcium phosphate with DIV6.

及び

を用いて逆PCRによって、pEGFP-LDLRに挿入した。EGFPを、プライマー

及びその逆補体を用いてQuikChange(商標) (Stratagene)により除去した。LDLR-Alaを、以前に開示されたプライマを用いてQuikChange(商標)によってLDLR-APから生じた(Chen, I.らの論文 (2005). Nat.Methods 2, 99-104；及びChen, I., Ting, A.Y. (2005). Curr.Opin.Biotechnol. 16, 35-40を参照されたい。その各々は全体において引用により取り込まれている。)。これらの変化は、DNA配列決定によって検査した。FH LDLR-APは、突然変異を含み、プライマー

及びその逆補体を用いてQuikchange(商標)により生じた。CMVbipep-neoのGluR2-APは、開示されている(Howarthらの論文, 2005)。pCIのポストシナプスマーカーHomer1b-GFPは、Yasunori Hayashi (MIT, US)の親切な贈り物であった。BirA-YFP-ERは構造：Igシグナル配列-HAタグ-BirA-YFP-KDELを有し、PCRによってE.コリBirA及び黄色蛍光タンパク質(YFP)をpDisplay (Invitrogen)に挿入し、かつQuikChange(商標)によってKDEL ER保持配列及びBirAの直後の停止コドンを挿入して生成した。 (Plasmid)
pEYFP-H2B was a kind gift from AK Leung (MIT, US). This encodes human histone H2B fused to EYFP as a nuclear local cotransfection marker. pcDNA3 enhanced blue fluorescent protein (a kind gift of RY Tsien) was used as a cytoplasmic co-transfection marker. LDLR-AP has AP and was inserted after the signal sequence of human LDLR, so that AP was exposed on the cell surface. LDLR (Clontech) from pEGFP was a kind gift from T. Kirchhausen (Harvard Medical School, US). AP, primer

as well as

Was inserted into pEGFP-LDLR by inverse PCR. EGFP, primer

And its reverse complement was removed by QuikChange ™ (Stratagene). LDLR-Ala was generated from LDLR-AP by QuikChange ™ using previously disclosed primers (Chen, I. et al. (2005). Nat. Methods 2, 99-104; and Chen, I Curr. Opin. Biotechnol. 16, 35-40, each of which is incorporated by reference in its entirety). These changes were examined by DNA sequencing. FH LDLR-AP contains mutations and primers

And its reverse complement using Quikchange ™. CMVbipep-neo's GluR2-AP has been disclosed (Howarth et al., 2005). The pCI post-synaptic marker Homer1b-GFP was a kind gift from Yasunori Hayashi (MIT, US). BirA-YFP-ER has the structure: Ig signal sequence-HA tag-BirA-YFP-KDEL, inserts E. coli BirA and yellow fluorescent protein (YFP) into pDisplay (Invitrogen) by PCR, and QuikChange (trademark) ) Inserted a KDEL ER retention sequence and a stop codon immediately after BirA.

及び

を用いてヒトEphA3のシグナル配列の直後に、逆PCRによって挿入した。pCIのヒト癌胎児性抗原(CEA)は、G. Prud'homme (University of Toronto, Canada)の親切な贈り物であった。pcDNA3のヒトEGFR-AP及びpDisplayのAP-CFP-TMは、開示されている(Chenらの論文, 2005)。 YFP was removed and BirA-ER was obtained by Quikchange ™. pEF-BOS EphA3-AP was a kind gift from Martin Lackmann (Monash University, Australia). AP, primer

as well as

Was inserted by reverse PCR immediately after the signal sequence of human EphA3. pCI's human carcinoembryonic antigen (CEA) was a kind gift of G. Prud'homme (University of Toronto, Canada). The pcDNA3 human EGFR-AP and the pDisplay AP-CFP-TM have been disclosed (Chen et al., 2005).

(Non-specific binding of nanocrystals)
HeLa cooled to 4 ° C in PBS for 5 min to minimize endocytosis, 4 ° C with 40 nM nanocrystals in PBS containing 0.5% dialyzed bovine N, N-dimethylcasein (Calbiochem) for 10 min Incubated for 10 minutes. Cells were then washed 4 times with ice cold PBS and imaged in PBS.

(Biotinylation and labeling of cells)
Cells were biotinylated in PBS 5 mM MgCl ₂ containing 2.6 μM biotin ligase and 10 μM biotin-AMP for 10 minutes at 24 ° C. (Howarth et al., 2006). Cells were then washed 4 times with PBS and incubated for 5 minutes at 24 ° C. with 20 nM nanocrystals in PBS containing 0.5% dialyzed bovine N, N-dimethylcasein (Calbiochem). Cells were washed 3 times with PBS and imaged in PBS at 24 ° C.

  For imaging of cell lines expressing BirA-ER, cells were incubated in normal growth medium (Tanabe USA) supplemented with 10 μM biotin 4 hours after transfection. The next day, the cells were washed 4 times with PBS and incubated for 5 minutes at 24 ° C. with 20 nM monovalent nanocrystals in PBS containing 0.5% dialyzed casein. For specificity testing, cells were prepared in PBS containing 3% dialyzed bovine serum albumin with 100 nM monovalent streptavidin Alexa Fluor 568 (prepared as disclosed in (Howarth et al., 2006)). Incubated for 10 minutes at 4 ° C. The cells were then washed 3 times with PBS and imaged live. For analysis of EphA3-AP cluster formation, cells were biotinylated as above for 10 minutes, washed 3 times in PBS, followed by 10 nM monovalent or multivalent nanocrystals (6 mSA: nanocrystals). The mixture was incubated at 37 ° C. for 14 minutes. Cells were washed 3 times in ice-cold PBS and imaged at 4 ° C. PBS was replaced with Tyrode's buffer for biotinylation and imaging of neurons.

(EGF receptor labeling with streptavidin linked to nanocrystals by EDC coupling)
COS7 cells were transfected with 0.2 μg pcDNA3 EGFR and 7.5 ng pcDNA3 BFP per well of a 48-well plate. The next day, cells in PBS containing 5 mM MgCl ₂ , 0.5% dialyzed casein and 90 nM biotinylated EGF (Biotin-XX-EGF from Invitrogen: human EGF linked to biotin via a long spacer arm at a single site), Incubated for 5 minutes at room temperature. Cells were washed 3 times with PBS, incubated with PBS, 0.5% dialyzed casein and 70 nM 20% amino NC605-wtSA for 5 minutes at room temperature, then washed 4 times with PBS and imaged in PBS. As a negative control, NC605-wtSA was incubated with a 500-fold excess of free biotin (Tanabe, USA) for 5 minutes at room temperature and then added to the cells. For video imaging, COS7 cells were labeled as described above except with 20 nM 20% amino NC605-wtSA and maintained in the microscope at 37 ° C. using an environmental management system (Solent Scientific). .

(FRET imaging of nanocrystals and dyes bound to EGF receptors)
A 5-fold molar excess of hSA in PBS was incubated with 20% amino NC558 or 20% amino NC558-Alexa Fluor 568 for 30 minutes at room temperature to stably bind the His ₆ -tag of hSA to the nanocrystal shell. . HeLa was transfected with 0.2 μg pcDNA3 EGFR and 5 ng H2B-YFP per well of a 48 well plate. The next day, cells are combined with PBS containing 5 mM MgCl ₂ , 0.5% dialyzed casein and 60 nM biotinylated EGF (Biotin-XX-EGF from Invitrogen: human EGF linked to biotin via a long spacer arm at a single site). Incubate to stop receptor internalization. Cells are washed 4 times with cold PBS, incubated with PBS, 0.5% dialyzed casein and 40 nM QD558-hSA or QD558-hSA-Alexa Fluor 568 for 5 minutes at 4 ° C, then washed 4 times with cold PBS for imaging did.

(Fluorescence and phase contrast microscopy)
Images of HeLa cells were collected in live cells using a Zeiss Axiovert 200M inverted photometrics with a 40x oil immersion lens and an intensified Cascade II camera set at 3500. YFP (495DF10 excitation, 515DRLP dichroism, 530DF30 emission), Alexa Fluor 568 (560DF20 excitation, 585DRLP dichroism, 605DF30 emission), QD 565 (405DF20 excitation, 515DRLP dichroism, 565DF20 emission), and QD 605 (405DF20 Excitation, 585DRLP dichroism, 605DF30 emission) images were collected and analyzed using Slidebook software (Intelligent Imaging Innovations). Typical exposure time was 0.1-0.5 seconds. AlexaFluor 568 fluorescence faded with strong 405DF20 excitation for 30 seconds. The fluorescence image was background corrected.

及び

(Eurogentec)を有するTaqポリメラーゼを用いて、95℃で30秒、50℃で30秒、72℃で1分間の27サイクルの条件により、PCRによって生成した。そのDNAを、TAE中、臭化エチジウム-染色2%アガロースゲルにおいて9.3V/cmで30分間泳動させた。唯一の119bp PCR産物をUV下で視覚化し、QIAquickゲル抽出キット(Qiagen)を用いてddH₂Oに抽出し、一定分量を−20℃で保存した。9nMビオチン化DNAを、10mMホウ酸ナトリウムpH 7.3中、3nMナノクリスタルとともに24℃で1時間インキュベートした。20μL 0.1%ポリ-L-リシン(Mw 500-2000)(Sigma)を、新たに切断されたマスコバイト雲母V2(Electron Microscopy Sciences)上にピペットで取った。3分間の培養の後、その雲母を1mLのddH₂Oで洗浄し、窒素流下で乾燥した。20μLのDNA-ナノクリスタル複合体を、その雲母上にピペットで取った。6分後に、その雲母を、1mLのddH₂Oでリンスし、窒素流下で乾燥した。その雲母を、273.3kHz共鳴周波数で作動する名目上＜10nm(Veeco Probes)のチップ半径を有するエッチングされたTESPシリコンプローブを使用して、タッピングモードでDimension 3100原子間力顕微鏡(Digital Instruments, Santa Barbara CA)を用いてイメージ化した。高さ及び位相画像を、2Hzの走査速度及び256×256ピクセルの解像度で、1×1μm域全体にわたって同時に収集した。得られたイメージを、Nanoscope v 5.30ソフトウェア(Digital Instruments)を用いて分析した。 (Nuclear microscope inspection)
Biotinylated DNA from pIVEX-BirA (a kind gift from A. Griffiths, MRC Laboratory for Molecular Biology, UK)

as well as

It was generated by PCR using Taq polymerase with (Eurogentec) under conditions of 27 cycles of 95 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 1 minute. The DNA was run on an ethidium bromide-stained 2% agarose gel in TAE for 30 minutes at 9.3 V / cm. A unique 119 bp PCR product was visualized under UV, extracted into ddH ₂ O using the QIAquick gel extraction kit (Qiagen) and aliquots stored at −20 ° C. 9 nM biotinylated DNA was incubated for 1 hour at 24 ° C. with 3 nM nanocrystals in 10 mM sodium borate pH 7.3. 20 μL 0.1% poly-L-lysine (Mw 500-2000) (Sigma) was pipetted onto freshly cut muscovite mica V2 (Electron Microscopy Sciences). After 3 minutes incubation, the mica was washed with 1 mL ddH ₂ O and dried under a stream of nitrogen. 20 μL of DNA-nanocrystal complex was pipetted onto the mica. After 6 minutes, the mica was rinsed with 1 mL ddH ₂ O and dried under a stream of nitrogen. The mica was measured using a Dimension 3100 atomic force microscope (Digital Instruments, Santa Barbara) in tapping mode using an etched TESP silicon probe with a nominal radius <10 nm (Veeco Probes) operating at a 273.3 kHz resonant frequency. CA). Height and phase images were collected simultaneously over the entire 1 × 1 μm area with a scan rate of 2 Hz and a resolution of 256 × 256 pixels. The resulting images were analyzed using Nanoscope v 5.30 software (Digital Instruments).

(Fluorescence correlation spectroscopy)
Two-photon fluorescence correlation spectroscopy measures the autocorrelation of the emission of nanocrystals in solution under a microscope objective and determines the time dynamics and thus the diffusion constant in the calibrated focal volume. As a result, the diffusion constant is related to the hydrodynamic diameter via the Stokes-Einstein relation. The homemade apparatus starts with 780 nm light from a Coherent Ti-Sapphire oscillator (Coherent Mira) pumped with an Ar ⁺ ion laser and operating at a pulse width of ˜120 fs and a repetition rate of 80 MHz. The light beam is sent through a 40x, 1.2NA immersion objective (Zeiss). Fluorescence is collected through a 720 longpass dichroic (Chroma) 400 nm holographic notch filter and the same objective filtered through the appropriate bandpass. As a result, the light passes through two avalanche photodiodes (EG & G and SPCM-AQR14) through a 50/50 beam splitter. The signals are cross-correlated by a multi-tau autocorrelator card (ALV GmbH). This cross-correlation reduces measurement noise and eliminates artifacts from photodiode afterpulses. The 3D focal volume waist and aspect ratio are calibrated according to the standard 3D Gaussian approximation. The aspect ratio is first calculated using known concentrations of R _H = 22 nm beads (Duke Scientific, product G40). This ratio is then applied to a series of newly filtered beads (R _H = 22 nm, 28.5 nm, 35.5 nm, 44 nm, Duke Scientific, G40, G50, G75, G80) to determine the beam waist. The R ² value for these agreements was greater than 0.999. We measured samples in the range of excitation forces to ensure that flashing, saturation and photobleaching were not under the regime that would affect the measurement (e.g., Larson, DR et al. Science 300 [5624], 1434-1436. 2003. It is incorporated by reference in its entirety. For final measurements, DHLA-PEG-CO ₂ H nanocrystals were excited at 0.75 mW at the back focal plane of the object filled with light, and commercially available nanocrystal-SA was excited at 0.5 mW. Samples were measured in 10 actuations each for 30 seconds and the r ² value was an average curve> 0.998. The hydrodynamic diameter was calculated as disclosed (see, eg, Schwille, P. et al. (1996). Biochemistry 35, 10182-10193, which is incorporated by reference in its entirety). .

(Antibody production)
A secretion vector for anti-CEA scFv sm3E has been disclosed (Graff, CP et al. (2004). Protein Eng Des Sel 17, 293-304, which is incorporated by reference in its entirety). To prevent protein dimerization, disulfide bonds were inserted between the VH and VL regions of scFv by mutating residues R44 and G234 to cysteine with QuikChange ™. In addition, cysteine was inserted into the C-terminus of scFv immediately before the His ₆ tag for PEGylation using QuikChange (trademark). The plasmid was changed to YVH10 yeast using the EZ yeast kit (Zymo Research) and seeded in SD-CAA medium supplemented with 40 μg / mL tryptophan. Individual colonies were grown in 1 L flasks and induced to secrete for 48 hours at 37 ° C. as disclosed (Graff et al., 2004). The clear supernatant was concentrated with a 10 kDa ultrafiltration membrane (Millipore) and His-tagged protein was purified with Talon metal affinity resin (BD Biosciences) following the manufacturer's batch-column protocol. Monomeric scFvs were further purified by size exclusion chromatography on a Superdex 75 column (GE Healthcare) and eluted in PEGylation buffer (100 mM Na ₂ HPO ₄ , 500 mM NaCl, 2 mM EDTA, pH 6.5). Unmodified scFv is too small (27 kDa) to give separated bands after ligation to nanocrystals, but PEGylation of scFv significantly increases effective hydrodynamic size without compromising antigen recognition Let For PEGylation, scFvs at a concentration of 0.5-1 mg / mL was added to a 5-fold molar excess of 5 kDa PEG-maleimide (Nektar) and immobilized Tris [2-carboxyl to reduce the gel at a 150 μL gel concentration per 1 mL reaction. Co-incubation with ethyl] phosphine hydrochloride (TCEP). The use of TCEP resin instead of soluble TCEP or dithiothreitol to reduce the C-terminal cysteine prevented the reduction of partially obscured disulfide bonds in the protein. The reaction mixture was incubated at 25 ° C. for 5 hours on a rocker. Thereafter, the TCEP resin was removed by centrifugation. Unreacted PEG was removed by ion exchange chromatography on a Hi-Q column (GE Healthcare) equilibrated with 20 mM Tris HCl (pH 8.2). PEGylated scFvs were separated from unlinked scFvs on a Superdex 75 column at a flow rate of 0.5 mL / min and eluted in PBS. The PEGylation efficiency and the purity of the conjugate were evaluated by SDS-PAGE.

(Ligand synthesis)
The diamine functionalized polyethylene glycol was synthesized in high yield from an inexpensive and commercially available PEG (average MW 400) starting material via a three step reaction with minimal required purification steps (Scheme 1). The overall yield was 89% on a 30 g scale. All intermediate products were easily monitored by FTIR spectroscopy.

After ligand exchange with compound 3 (DHLA-PEG-NH ₂ ), the nanocrystals become water dispersible and undergo further covalent modification via simple N-hydroxysuccinimidyl (NHS) coupling chemistry Had surface amino groups that could be

Alternatively, compound 2 was further modified to give compound 6 (DHLA-PEG-CO ₂ H), which gave nanocrystals with water-dispersible carboxyl surface groups after ligand exchange.

  The synthetic scheme shown has a multidentate coordination site at one end for strong coordination to the nanocrystal surface, a short spacer that transmits water solubility and reduces non-specific binding, and further derivatization at the end. It provides a simple means for heterobifunctional ligands with ionizing functionality that allow. Furthermore, all coupling can be achieved through amide bond formation, which is more stable than hydrolysis to ester bonds. This feature can be particularly relevant for stability in cell labeling where the cellular environment can contain many non-specific esterases (eg, Gerolf, VZ, ACTA Path. Micro. IM. B. 1970, 78, 258- (See 260, which is incorporated by reference in its entirety.)

(Ligand exchange and characterization of functional hydrophilic nanocrystals)
Nanocrystals emitting at 565 and 600 nm were synthesized in several variations according to well-known literature procedures. Briefly, Cd and Se precursors were rapidly injected into a hot solvent system to form a CdSe core. Subsequently, the core was purified by precipitation, redispersed in a coordinating solvent, and overcoated with about five single layer alloys Zn _x Cd _(1-x) S shell (x≈0.7). As shown in FIG. 1A, the formation of the alloy shell was accompanied by a red shift as great as 30 nm in absorbance at the shell growth. FIG. 1A shows NC605 absorption spectra of a CdSe core (black) and an overcoated CdSe (Zn _x Cd _(1-x) S)) core (shell) (red). Fluorescence spectrum of NC605 in hexane excited at 520 nm after 1 cycle precipitation (green, QY = 65%), and fluorescence spectrum of NC605 in PBS buffer excited at 520 nm after ligand exchange with compound 3. (Blue, QY = 43%) indicates the smallest decrease.

The use of an alloy shell has been found to enhance the quantum yield of nanocrystals by a factor of 2 in migration to aqueous solution compared to nanocrystals overcoated with pure ZnS shell. Ligand exchange of CdSe (Zn _x Cd _(1-x) S) core (shell) nanocrystals using DHLA-PEG-NH ₂ (compound 3) and DHLA-PEG-CO ₂ H (compound 6) Performed according to previously reported procedures (see, eg, Uyeda, HT et al., J. Am. Chem. Soc. 2005, 127, 3870-3878, which is incorporated by reference in its entirety. ). Briefly, 200 μL of nanocrystals in the growth solution was precipitated by addition of polar solvent to obtain ˜10 mg of dry pellets, and 50 μL of neat DHLA-PEG derivatized ligand and 10 μL of MeOH were added. The mixture was gently stirred at 60 ° C. under N ₂ for 2.5 hours and precipitated by the addition of ethanol, chloroform and hexane. Centrifugation at 3000 g for 2 minutes gave a clear supernatant containing excess ligand and organic soluble impurities, which was discarded. The pellet was dispersed in 0.5 mL phosphate buffered saline (PBS, pH 7.4), filtered through a 0.2 μm filter to remove aggregates, and purified by two cycles of ultrafiltration. The typical QY of nanocrystals capped with DHLA-PEG-NH ₂ (compound 3) is 33%, while the QY of nanocrystals capped with DHLA-PEG-CO ₂ H (compound 6) is water-soluble. It was about 43% after solubilization.

The HD of nanocrystals capped with DHLA-PEG-CO ₂ H was measured by dynamic light scattering (DLS) and found to be ˜11.2 nm for NC605. See FIG. 1B showing representative DLS data for NC605 ligand exchanged with Compound 3 in PBS, with HD = 11.2 nm. As expected, the HD of the same nanocrystal ligand exchanged with Compound 3 or Compound 6 showed approximately the same HD of about ˜11 nm due to the comparable size of the ligand coating.

  The stability of nanocrystals ligand exchanged with Compound 3 or Compound 6 was tested in aqueous buffer ranging from pH 5 to pH 9. After 2 days under ambient laboratory conditions, the nanocrystals remained well dispersed in the solution. It highlights the excellent pH stability of nanocrystals coated with DHLA-PEG-based ligands consistent with previous reports.

(Adjustment of surface charge and functional group valence using mixed ligand system)
Nanocrystals ligand-exchanged with Compound 3 or Compound 6, respectively, have surfaces that are positively and negatively charged in neutral buffer, resulting in nonspecific binding despite the passivated PEG spacer. Can be involved. The surface charge is adjusted by ligand exchange using a mixture of compound 3 or compound 6 and neutral DHLA-PEG ₈ -OH in addition to the ratio of amine or carboxy functional groups on the nanocrystal surface. Can be synthesized according to previously reported procedures. The concept of ligand exchange using a mixture of different DHLA-based ligands is described by Uyeda, HT et al., J. Am. Chem. Soc. 2005, 127, 3870-3878, which is incorporated by reference in its entirety. First shown. Similarly, the present inventors performed mixed exchange by ligand exchange using DHLA-PEG-NH ₂ or a mixture in which the ratio of DHLA-PEG-CO ₂ H and DHLA-PEG-OH was changed. A ligand aqueous nanocrystal was prepared. The surface charge of the resulting nanocrystals was analyzed by zeta potential and agarose gel electrophoresis (see FIGS. 2A and 2B). Measurements show that nanocrystals simply capped with DHLA-PEG-NH ₂ have a zeta potential of +36.2 mV and decrease in a nearly linear fashion with decreasing -NH ₂ composition in the ligand mixture. It was. A similar trend was observed with carboxy-coated nanocrystals (Figure 2A) and was reflected in electrophoretic gel mobility (Figure 2B). Nanocrystals coated with 100% DHLA are also included in the measurement for comparison, and DHLA-capped nanocrystals have a higher ligand packing density and / or a smaller hydrodynamic radius, so 100% It was shown to have a higher negative charge than DHLA-PEG-CO ₂ H coated nanocrystals.

Ligand exchange using a mixed ligand system adjusts the surface charge and controls the valence per particle of functional groups on the nanocrystal surface. The number of reactive functional groups per nanocrystal was investigated by exposing amine-coated nanocrystals to the fluorogenic probe fluorescamine, which rapidly reacts with primary amines to form a fluorescent product that can be monitored at 487 nm. Nanocrystals ligand exchanged with various mixtures of -NH ₂ and -OH functionalized DHLA-PEG are repeatedly purified by ultrafiltration to remove excess ligand and react with excess fluorescamine. It was. Obtained after the reaction with the 3 samples at known concentration, by calibrating fluorescence intensity of fluorescamine, an estimate of 140 amines / nanocrystal for 100% of the DHLA-PEG-NH ₂ ligand exchange nanocrystals It was. This is consistent with previously reported values obtained using a slightly different method (Ballou, B. et al., Bioconjugate Chem. 2004, 15, 79-86, which is incorporated by reference in its entirety. .) Furthermore, a nearly linear correlation was obtained with respect to the proportion of amine-terminated ligand used in the cap-exchange mixture and the amount of amine detected per nanocrystal.

  These results show that the use of a mixed ligand mixture of charged and neutral DHLA-PEG-based ligands is a simple and powerful way to accurately adjust the final surface composition and charge of the resulting water-soluble nanocrystals Suggested to provide. Such a mixed ligand system is such that the nanocrystal exhibits a high enough valency surface amino or carboxyl group per particle for the application, while minimizing the charge that can reduce its non-specific binding. Means can be provided for maintaining the particle surface.

(Evaluation of non-specific cell binding)
The problem of non-specific binding must be addressed for nanocrystals useful for cell labeling applications, especially for single molecule imaging where high signal-to-noise ratio is the limit to obtain meaningful data. Don't be. In nanocrystal imaging experiments, nonspecific cell binding is due in part to electrostatic interactions on the cell surface with a charged nanocrystal ligand coating, and as a result of incomplete ligand exchange, It may also be caused by hydrophobic interactions between the lipid and the remaining hydrophobic capping ligand on the nanocrystal surface. See, for example, Bentzen, EL et al., Bioconjugate Chem. 2005, 16, 1488-1494. It is incorporated by reference throughout. ). Nanocrystals coated with various ligands were exposed to human cell lines at 4 ° C. for 10 minutes. The cells were then washed with buffer to remove unbound nanocrystals and the remaining nanocrystal fluorescence was imaged to investigate the effect of nonspecific binding as a function of the ligand composition. See Figure 3.

All fluorescence images in FIG. 3 were obtained under the same camera conditions and were displayed at the same contrast level so that they could be directly compared. The control sample (FIG. 3A) showed luminescence from cell autofluorescence. Nanocrystals coated with negatively charged DHLA showed significant non-specific binding to cells and glass (FIG. 3B). Neutral DHLA-PEG-OH coated nanocrystals showed the expected minimal non-specific binding (FIG. 3C). In this case, cell autofluorescence was clearly seen. Several individual nanocrystals that are non-specifically bound are indicated by arrows. Surprisingly, DHLA-PEG-CO ₂ H-coated nanocrystals, which have a similar charge as DHLA-coated nanocrystals as shown by agarose gel shift, are also non-specific seen in neutral PEG-OH functionalized nanocrystals. It showed minimal non-specific cell binding compared to cell binding (FIG. 3D). The lack of non-specific binding of DHLA-PEG-CO ₂ H-coated nanocrystals despite the negative charge highlights the important role of PEG spacers in reducing non-specific binding (FIG. 3C). However, when ligand exchange was performed with a DHLA-PEG-CO ₂ H sample in which the dihydrolipoate moiety was partially oxidized to its ring-closed form within a few days under ambient conditions, The degree of binding increased. This increase in non-specific binding was due to a decrease in the efficiency of ligand exchange. Probably results in incomplete replacement of the first hydrophobic capping shell. For minimal non-specific binding, it was important to store DHLA compounds under light shielding, <4 ° C., under N ₂ atmosphere to minimize oxidation.

On the other hand, DHLA-PEG-NH ₂ coated nanocrystals showed terrible non-specific binding to the cell membrane (data not shown). This was due to electrostatic interactions between strongly positively charged nanocrystals and negatively charged cell membranes. However, -OH functionalized DHLA-PEG nanoparticles were coated with a 20% mixture of -NH ₂ functionalized DHLA-PEG with respect to the crystal (Fig. 3E) is a non-specific binding than the -OH functionalized DHLA-PEG alone Not shown significantly.

These results show that DHLA-PEG-CO ₂ H and 20% DHLA-PEG-NH ₂ coated nanocrystals show minimal non-specific binding to cells, making them suitable for cell labeling and single particle imaging applications Suggests that In addition, 20% DHLA-PEG-NH ₂ coated nanocrystals show the importance and versatility of ligand exchange using a mixed ligand system to match the surface properties of the nanocrystals to the desired application.

(Covalent linkage to dye for FRET detection)
To evaluate the suitability of mixed amine / hydroxyl -PEG capped nanocrystals for routine derivatization, nanocrystals coated with 80% -OH and 20% -NH ₂ terminus ligand, the red light emitting organic It was reacted with an amine-reactive N-hydroxysuccinimidyl ester of the fluorescent dye carboxy-X-rhodamine (ROX) (FIG. 4A). The spectral overlap between nanocrystal photoluminescence and dye absorbance results in efficient Forster resonance energy transfer (FRET) from the nanocrystal to the dye. As such, this system serves as a good model for a recently investigated chemically sensitive nanocrystal-dye energy transfer system (Snee, PT et al., J. Am. Chem. Soc. 2006, 128, 13320 -13321, which is incorporated by reference in its entirety.)

  After reaction with ROX succinimidyl ester, the nanocrystals were separated from unbound dye and NHS byproduct via ultrafiltration. UV-Vis absorption spectra were used to monitor the dye: nanocrystal molar ratio before and after purification. FIG. 4B shows the absorption spectra of the starting nanocrystal, reaction mixture and purified product with clearly visible dye absorption peaks. Spectral agreement as the sum of nanocrystal and dye contributions represents a dye: nanocrystal ratio of 5.0 on mixing and 2.9 on purification, which represents a coupling yield of 58%. In a control experiment (FIG. 4C), nanocrystal samples from the same batch were mixed with the free acid form of ROX under the same reaction conditions. Less than 3.4% dye remained upon purification. As expected, primary amines with free carboxylic acids would not react under the mild conditions of the ligation procedure.

The FRET efficiency of the nanocrystal-dye pair was estimated to be ˜90% by measuring the amount of nanocrystal fluorescence quenching after dye ligation. From the measured FRET efficiency, the measured amount of dye per nanocrystal and the Forster distance (R ₀ = 5.6 nm), the separation distance was calculated to be r = 4.8 nm (e.g. Aaron R. Clapp (See ChemPhysChem 2006, 7, 47-57, which is incorporated by reference in its entirety). The purified nanocrystal-ROX conjugate was further characterized by gel filtration chromatography (GFC) using in-line detection of absorbance at 280 nm and acquisition of full spectrum fluorescence excited at 460 nm. Comparison with known hydrodynamic diameter (HD) protein molecular weight standards (FIG. 4D) provided an indication of size for the sample under investigation. FIG. 4E shows the GFC results for the purified nanocrystal-ROX conjugate shown in FIG. 4B. A single UV absorption feature was detected at an elution volume of 15.7 mL, corresponding to a protein equivalent molecular weight of ˜94 kD or HD of ˜8.2 nm, reasonably consistent with HD obtained by DLS. The emission spectrum at this amount showed two prominent features at 558 nm and 610 nm, corresponding to the nanocrystal and ROX emission wavelengths, respectively. The presence of ROX emission at the elution volume corresponding to the nanocrystal indicates that the nanocrystal and the dye are actually bound.

  In contrast, a GFC of a mixture of free nanocrystals and ROX dye at the same dye: nanocrystal ratio as the bound sample showed UV absorbance and nanocrystal signal at the same elution volume as the conjugate, but no dye fluorescence. (FIG. 4F). In fact, no dye was detected at the absorbance and excitation wavelength used by the instrument due to its low absorption cross section: ROX fluorescence is bound because the dye molecules bound to the nanocrystal are excited by FRET. Detected in the cases. Also, in this experiment, unbound nanocrystals eluted at 15.7 mL, suggesting that dye binding did not significantly alter nanocrystal HD. Overall, GFC data show that NHS ester-type small molecule dyes can be covalently bound to mixed ligand nanocrystals without disrupting nanocrystal size, and control experiments with non-activated dyes are coupled / coupled. Showed no signs of.

(Covalent binding to streptavidin for high affinity cell labeling)
A labeled construct was designed in which the streptavidin / biotin interaction was solved to directly link the nanocrystal to the membrane protein. This method avoided the need for bulky intermediate primary and secondary antibodies (see, eg, Wu, X. et al., Nature Biotechnol. 2003, 21, 41-46, which is incorporated by reference in its entirety. ing.). Direct labeling was made possible by the development of biotin ligase (BirA) for site-specific biotinylation of a 15 amino acid recognition sequence called a receptor peptide fused to either end of the receptor of interest (Figure 5). ; Howarth, M. et al., PNAS. 2005, 102, 7583-7588, which is incorporated by reference in its entirety. By adding recombinant BirA, biotin and ATP to the cell culture medium, the AP tag can be specifically and efficiently biotinylated, and then nanocrystals (nanocrystal-SA) linked to streptavidin, It can be applied to visualize biotinylated protein populations.

Therefore, SA was linked to 20% -NH ₂ /80% -OH DHLA-PEG coated nanocrystals. First, SA was activated in MES buffer (pH 5.5) with 1000 equivalents of EDC / NHS for 30 minutes. In order for the coupling to proceed efficiently, it was important to remove excess coupling reagent by ultrafiltration before applying the activated SA to the amine-functionalized nanocrystals. Activated SA was mixed with nanocrystals in bicarbonate buffer (pH 8.4), reacted for 1 hour, and purified by ultrafiltration. Nanocrystal-SA was applied to HeLa cells transfected with low density lipoprotein receptor-receptor peptide fusion construct (LDLR-AP) with enhanced yellow fluorescent protein (EYFP) as a nuclear co-transfection marker. Therefore, cells that show EYFP fluorescence from the nucleus also contain LDLR-AP on the cell surface. Nanocrystal-SA was applied to the cell culture medium at 50 nM after biotinylation of LDLR-AP, and specific binding of nanocrystals was observed on the surface of the transfected cells (FIG. 6A). Adjacent untransfected cells (indicated by the lack of EYFP nuclear markers) did not show nanocrystal-SA binding, revealing high specificity of labeling. In addition, the control experiment (FIG. 6B) using unlinked nanocrystals and the control experiment (FIG. 6C) excluding BirA showed no binding. In FIG. 6, red indicates NC605 channel; green, EYFP channel. FIG. 6A is an image of AP-LDLR receptor on HeLa cells biotinylated with BirA and labeled with streptavidin covalently attached to 20% -NH / -OH DHLA-PEG coated nanocrystals. 6B and 6C are a control experiment using unbound nanocrystals and a control experiment without BirA, respectively.

To study the mobility of monovalent nanocrystals in mutants of low density lipoprotein (LDL) receptors at the deleted cytosolic end, originally found in individuals with familial hypercholesterolemia (FH), (See, eg, Hobbs, HH et al., Annu. Rev. Genet. 24, 133-170 (1990), which is incorporated by reference in its entirety). The FH phenotype has been extensively analyzed according to the ligand of the receptor, LDL, but this method analyzed the behavior of the receptor itself. To confirm fast internalization of wt AP-tagged receptors, a pulse chase using mSA-Alexa Fluor 568 was compared to FH. Single monovalent nanocrystals bound to biotinylated AP-LDL receptor were imaged as indicated by nanocrystal fluorescence intensity and flashing. The mobility of receptors labeled with monovalent nanocrystals is significantly greater with respect to FH than wild-type LDL receptor (p = 1.6x10 ^-14 ) (Figure 6D), due to clathrin-coated pits adapters. Consistent with the binding of the wild-type cytosolic end (see, eg, Michaele, P. et al., J. Biol. Chem. 279, 34023-34031 (2004), which is incorporated by reference in its entirety. .).

FIG. 6D shows the results of single molecule tracking of the LDL receptor using monovalent nanocrystals. The histogram shows different distributions of single molecule diffusion coefficients for wild type and FH LDL receptors. (Kolmogorov-Smirnov D ′ statistic = 0.317, one side, p = 1.6 × 10 ⁻¹⁴ ) Mean log (D), −1.36 (416 tracks) for WT. About FH, -0.84 (256 tracks).

(His ₆ -tag binding of protein)
DHLA-PEG-CO ₂ H coated nanocrystals showed high mobility on agarose gels. We incubated these nanocrystals with a His _6- tagged monovalent variant of streptavidin (mSA) containing a single biotin binding site (e.g. Howarth, M. et al., Nat Meth 2006 , 3, 267-273, which is incorporated by reference in its entirety.) A significant decrease in the gel mobility of the nanocrystals was observed (FIG. 7A). Probably because of the metal-affinity driven self-assembly of proteins on the nanocrystal surface (nanocrystal-mSA). See, for example, Pons, T. et al., J. Phys. Chem. B 2006, 110, 20308-20316. It is incorporated by reference throughout. Incubation with wild type streptavidin (wtSA) did not feature a His ₆ -tag and there was no change in nanocrystal mobility on the gel (FIG. 7A). To demonstrate that the linking protein retained biological functionality, nanocrystal-mSA was tested in cell labeling. Addition of nanocrystal-mSA to HeLa cells showing biotinylated AP-LDLR resulted in a high degree of specific binding (FIG. 7B). All control experiments that attempted labeling with unbound nanocrystals (Figure 7C), nanocrystals incubated with wtSA (Figure 7D), and nanocrystal-mSA without addition of BirA (Figure 7E) were all as expected, No binding occurred. Furthermore, the stability, specificity and high QY of these nanocrystals allowed single particle tracking of LDLR on the cell surface over time.

Linkage to mSA was achieved simply by mixing the desired ratio of nanocrystals to His _6- tagged protein followed by 1 hour incubation at room temperature. No coupling agent or purification step was required to purify nanocrystals suitable for cell labeling. The ease of this linking method is emphasized in biological functional nanocrystals for targeted cell imaging applications. Furthermore, the unique property of nanocrystal gel shift after His ₆ -tag protein ligation is that a given ratio of mSA to nanocrystal results in a ladder of bands according to the Poisson intensity distribution. Each band represents a nanocrystal bound to a different number of mSA (e.g. Pons, T .; Uyeda, HT; Medintz, IL; Mattoussi, H., J. Phys. Chem. B 2006, 110, 20308- (See 20316, which is incorporated by reference in its entirety.) True monovalent nanocrystals can be prepared if the first band corresponding to nanocrystals bound to exactly one mSA can be separated. A single biotin binding site of mSA, with a single copy of mSA per nanocrystal, reduces the amount of binding sites per nanocrystal to exactly one, and contains up to 20 wtSA per nanocrystal, thus 1 nanocrystal Eliminate any possibility of cross-linked protein target, a major problem with commercially available nanocrystals containing up to 80 binding sites per hit. See, for example, Medintz, I. et al., Nature Mater. 2005, 4, 435-446. It is incorporated by reference throughout.

(Application of shared and non-covalent strategies)
First, NC565 coated with 20% -NH ₂ /80% -OH DHLA-PEG was linked to Alexa 568 NHS-ester via amide bond formation. After purification by ultrafiltration, the average number of dyes for the nanocrystals was calculated to be ˜1.3 from the UV-vis absorbance spectrum. Excitation at 420 nm with minimal dye absorbance resulted in nanocrystal and dye emission, suggesting the occurrence of FRET. Based on quenching of nanocrystal fluorescence compared to nanocrystals without bound dye, the FRET efficiency was estimated to be ˜74%. The nanocrystal-dye conjugate was then incubated with mSA and applied to HeLa cells showing biotinylated AP-LDLR for labeling (FIGS. 8A-8B). Two emissions from nanocrystals and dyes were seen in the green and red channels, respectively, and the coexistence of the two channels on the target cells examined the integrity of the nanocrystal-dye-mSA conjugate (Figure 8B). . In response to intensity irradiation with long-term excitation at 420 nm, dye fading was observed in the red channel along with the recovery of nanocrystal emission in the green channel. This further suggests the generation of FRET from the targeted nanocrystal-dye on the cell surface. Control samples using nanocrystal-mSA without bound dye did not differ in red and green channel ratios after prolonged irradiation.

(Characteristics of monovalent nanocrystals)
To confirm the valence of the isolated nanocrystals, we performed atomic force microscopy (AFM) on monovalent nanocrystals incubated with a 3-fold excess of monobiotinylated DNA (Fig. 11A). AFM visualized nanocrystals and single molecules of DNA, showing nanocrystals bound to single biotinylated DNA. This supported the presence of a single biotin binding site per nanocrystal. When nanocrystals linked by several copies of monovalent streptavidin (multivalent nanocrystals) were analyzed in the same way, multiple copies of DNA bound to the nanocrystals. Multivalent nanocrystals appeared larger in AFM as a result of the mSA extra copy contributing to nanoparticle size.

  In addition, nanocrystals bound to a single copy of the monovalent antibody fragment were purified by agarose electrophoresis. See FIG. 10B. Single chain Fv antibodies selected by yeast surface display that bind exceptionally strongly to the tumor marker carcinoembryonic antigen were selected (Graff et al., 2004). Monovalent antibody-nanocrystals were similarly purified (FIGS. 13A-13B) and specifically labeled carcinoembryonic antigen was expressed on the cell surface (FIG. 13C). Monovalent antibody-nanocrystals did not label cells that were instead transfected with LDLR-AP, and unlinked nanocrystals did not bind carcinoembryonic antigen. The protein needed to be> 50 kDa to separate nanocrystal conjugates according to valence. Also, electrophoretic separation of nanocrystals according to valence was efficient for commercially available polyacrylic acid-coated nanocrystals, but these nanocrystals gave an unacceptably high nonspecific adhesion to the cells. As previously observed, PEG-amine coated nanocrystals had low non-specific binding, but not enough migration on the gel to be separated according to valence. See, for example, So, M.K. et al. (2006). Nat. Biotechnol. 24, 339-343. It is incorporated by reference throughout.

(Transferred BirA-ER allows targeting of monovalent nanocrystals.)
Receptor peptide-tagged proteins on the surface of living cells were biotinylated by adding recombinant biotin ligase to the medium (Howarth et al., 2005; Chen et al., 2005). Targeting cells to the endoplasmic reticulum (ER) with an immunoglobulin signal sequence to simplify the labeling of receptors with streptavidin-nanocrystals for cell biology and potential applications in living animals And was retained in the endoplasmic reticulum by the KDEL C-terminal sequence (BirA-ER). Biotin ligase expressed in the secretory pathway has previously been used for gene therapy and antibody modification applications, not for receptor tracking. For example, Nesbeth, D. et al. (2006). Mol. Ther. 13, 814-822; and Barat, B., Wu, AM (2007). “Recombinant antibodies by biotin ligase retained in the endoplasmic reticulum. See Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum. Biomol. Eng. Each of which is incorporated by reference in its entirety.

  BirA-ER can efficiently biotinylate AP condensed to low density lipoprotein receptor (LDLR) and label the cell surface with streptavidin-dye (FIGS. 9B and 11B). BirA-ER did not biotinylate endogenous cell surface proteins. This is because cells are not stained with streptavidin when BirA-ER is expressed with a negative control construct having a variant of AP (LDLR-Ala) (FIG. 11B). This method was generally effective: BirA-ER was also used to label EphA3 condensed to epidermal growth factor receptor and AP of HeLa cells and GluR2-AP of neurons (FIG. 15B). This outline contrasts with previous reports of receptor biotinylation by cytosolic biotin ligase. It only biotinylates a subset of biotin receptor regions. Normal growth medium biotin (˜9 nM) may be sufficient for biotinylation of AP fusions in the ER (eg, Baumgartner, MR et al. (2004). Am. J. Hum. Genet. 75, 790 See -800, which is incorporated by reference throughout.) However, supplementing the growth medium with biotin on the day of transfection can improve biotinylation efficiency. 10 μM biotin gave optimal biotinylation of LDLR-AP. Biotin did not produce an apparent change in the viability of HeLa or neuron cultures, even at 1 mM. For neurons, it was not necessary to supplement the medium with biotin to obtain efficient AP biotinylation with BirA-ER (FIG. 13).

  Monovalent nanocrystals were used to label LDLR-AP biotinylated by endoplasmic reticulum BirA (FIG. 11B). In this case, we used a variant of BirA-ER including the yellow fluorescent protein (YFP) BirA-YFP-ER. It behaved similarly to BirA-ER, but also allowed visualization of transfected cells. Monovalent nanocrystals bound only to AP-expressing cells and did not bind to LDLR-expressing negative control cells (LDLR-Ala) having AP tag point mutations. This indicated that monovalent nanocrystals retain a high specificity of cell labeling, similar to commercially available streptavidin-nanocrystals (Howarth et al., 2005).

(Nanocrystal monovalence avoids obstacles to receptor activation and mobility)
Receptor clustering is a common method of activating signal transduction. On the other hand, nanoparticles used for single particle tracking are often multivalent (eg, Saxton, MJ, Jacobson, K. (1997). Annu. Rev. Biophys. Biomol. And Jaiswal, JK and Simon, SM Trends Cell Biol. 14 [9], 497-504. 2004, each of which is incorporated by reference in its entirety. The tyrosine kinase EphA3, a receptor for ephrin that has an important role in developmental and metastatic cell motility, was phosphorylated when clustered by ephrin-coated beads. See, for example, Wimmer-Kleikamp, SH, Lackmann, M. (2005). IUBMB. Life 57, 421-431; and Wimmer-Kleikamp, SH et al. (2004). J. Cell Biol. 164, 661-666. I want to be. Each of which is incorporated by reference in its entirety. Valence-controlled nanocrystals were used for imaging EphA3-AP expressing CHO cells. EphA3-AP was incubated with DHLA-PEG8-CO ₂ H nanocrystals conjugated to one or more copies of monovalent streptavidin (FIG. 12). Monovalent nanocrystals labeled the receptor and maintained broad localization on the cell surface. This pattern of staining was the same as when EphA3-AP was labeled with monovalent streptavidin linked to Alexa Fluor 568 dye. In contrast, multivalent nanocrystals clustered EphA3-AP and induced internalization, as observed with ephrin bead stimulation. Commercial (multivalent) streptavidin-nanocrystals similarly produced receptor cluster formation and internalization.

  Receptor cross-linking also caused a decrease in receptor mobility on the receptor cell surface. We investigated the mobility of low density lipoprotein receptor (LDLR) mutants at the deleted cytosolic end, initially found in individuals with familial hypercholesterolemia (FH). In the form of low density lipoprotein, LDLR is a key receptor for cholesterol uptake by the periphery. This deletion mutant abolishes the retention of coated pits and reduced endocytic receptors. LDLR has been previously imaged at the single molecule level by labeling its ligand LDL. If the ligand-bound and unbound receptors have the same transport behavior, it is not yet clear. Receptor FH LDLR-AP was biotinylated with exogenous biotin ligase and labeled by adding monovalent or multivalent nanocrystals. Single receptor-bound nanocrystals can be individually imaged as shown by nanocrystal fluorescence intensity and flashing (see, eg, Nirmal, M. et al. (1996). Nature 383, 802-804. See, it is incorporated by reference in its entirety.) The mobility of receptors labeled with monovalent nanocrystals was substantially greater than those labeled with multivalent nanocrystals, consistent with receptor cross-linking with multivalent nanocrystals.

(The monovalent nanocrystal has the same size as the antibody.)
For many cell locations, the size of the nanoparticles bound to the receptor does not significantly affect the receptor mobility. For example, Borgdorff, AJ, Choquet, D. (2002) Nature 417, 649-653; Kusumi, A. et al. (2005). Annu. Rev. Biophys. Biomol. Struct. 34, 351-378; and Thoumine, See O. et al. (2005) Biophys. J. 89, L40-L42. Each of which is incorporated by reference in its entirety. Transmembrane helices and interactions at the cytosolic end are often determinants of receptor mobility (Saxton and Jacobson, 1997). However, due to the specific limited position, the nanoparticle size can be significantly delayed in mobility or can be confused at labeling, especially at neuronal synapses only 24-30 nm wide. For example, Groc, L. et al. (2004). Nat. Neurosci. 7, 695-696; Howarth, M. et al. (2005). Proc. Natl. Acad. Sci. USA 102, 7583-7588; See Zuber, B. et al. (2005). Proc. Natl. Acad. Sci. USA 102, 19192-19197. Each of which is incorporated by reference in its entirety.

DHLA-PEG8-CO ₂ H nanocrystals emitting at 605 nm gave a hydrodynamic diameter of 11 nm. This is an IgG antibody (9.7 nm), a standard probe for cell surface labeling by dynamic light scattering (DLS, Table 1) and a probe used with single particle tracking but with limited light stability. Determined relative to unlinked R-phycoerythrin (11 nm). Commonly used commercially available streptavidin nanocrystals emitting at 605 nm have a diameter of 21 nm. The generation of nanocrystals bound to a single monovalent streptavidin rather than multiple copies must contribute to keeping the size as small as possible. The inventors have found that the diameter increases by 1.2 nm in response to the binding of a single monovalent streptavidin to the nanocrystal. Another measurement method for nanocrystal size, fluorescence correlation spectroscopy (FCS), gave comparable values (Table 1).

(Small nanocrystals improve synaptic staining of AMPA receptors)
Reducing the size of the streptavidin-nanocrystal from 21 nm to 12 nm improved nanocrystal access to synapses because it labeled the AMPA receptor subunit GluR2. Dissociated cultured hippocampal neurons are GluR2-AP, a synaptic marker Homer1b-GFP (see Xiao, B. et al. (1998). Neuron 21, 707-716, which is incorporated by reference in its entirety). , And BirA-ER. GluR2-AP was biotinylated with BirA-ER and the surface was labeled with monovalent nanocrystals or commercially available streptavidin nanocrystals. Thereafter, the coexistence of nanocrystals and synapses was compared (FIG. 13). As previously observed (Howarth et al., 2005), commercially available nanocrystals were present in large areas outside synapses. However, monovalent nanocrystal labels aggregated at synapses. Consistent with the reduced size of these nanocrystals, the ability to label GluR2 accurately was improved (see, eg, Passafaro, M. et al. (2001). Nat. Neurosci. 4, 917-926). It is incorporated by reference throughout.)

(EGFR tracking)
Amino NC-SA conjugates were applied for specific cell labeling of EGF receptor (EGFR) on live cells and single particle tracking. EGFR is an important activator of cell division and is a target for many cancer therapies (see, eg, Moasser, MM, Oncogene 2007, 26, 6577-92, which is incorporated by reference in its entirety. .) There are still many questions about the mechanism of receptor association and internalization in EGFR signaling. They are optimally addressed through studies at the single molecule level. See, for example, Lidke, DS et al., J. Cell. Biol. 2005, 170, 619-626, and Teramura, Y. et al., EMBO J. 2006, 25, 4215-22. Each of which is incorporated by reference in its entirety. ).

  FIG. 16 schematically shows the target of live cells transfected with human EGFR. COS7 cells were incubated with biotinylated EGF (bioEGF) and then stained with amino NC-SA. Nanocrystals were observed to specifically bind to the surface of EGFR transfected cells, as indicated by the blue fluorescent protein (BFP) co-transfection marker. See the left panel of FIG. It shows the targeting of 20% amino NC-SA conjugates to EGFR on living cells. EGFR expressing cells are indicated by BFP cotransfection markers. Upper row: NC558 channel. Bottom row: BFP + DIC channel. Left column: EGFR transfected COS7 cells treated with biotinylated EGF and stained with amino NC-SA. Right column: Control experiment in which amino NC-SA was prevented by excess free biotin. Scale bar, 10 μm. Adjacent untransfected cells indicated by the absence of the BFP marker did not show nanocrystal staining, indicating the specificity of labeling.

Furthermore, the photostability, specificity and high QY of these nanocrystals allowed single particle tracking of EGF interaction with EGFR on the cell surface (Figure 18 left: DIC channel. Right: NC605. Channels with high brightness show autofluorescence, and dots represent clusters of nanocrystals (scale bar, 5 μm). Individual nanocrystals could be identified by their fluorescence intensity and intermittent, or blinking, behavior, and could be seen moving on the cell surface in a manner consistent with the active transport of labeled receptors.
Other embodiments are within the scope of the appended claims.

Claims (26)

  A method for producing a valence controlled semiconductor nanocrystal comprising: contacting a population of semiconductor nanocrystals with a compound having an affinity for the semiconductor nanocrystal to form a distribution of compound-bound nanocrystals; and Separating the members of the distribution of compound-bound nanocrystals according to the number of compounds bound to each nanocrystal.   The method of claim 1, further comprising isolating a member of the distribution of compound-bound nanocrystals wherein exactly one compound is bound to the nanocrystals.   The method of claim 2, wherein the compound is capable of selectively binding a ligand.   4. The method of claim 3, wherein the compound is capable of selectively binding exactly one ligand.   3. The method according to claim 2, wherein the compound is avidin or streptavidin.   6. The method of claim 5, wherein the compound is monovalent avidin or monovalent streptavidin.   6. The method of claim 5, wherein the compound comprises a polyhistidine tag.   4. The method of claim 3, wherein the compound is an antibody.   9. The method of claim 8, wherein the compound is a single chain antibody. The method of claim 1, wherein the semiconductor nanocrystal comprises an outer layer comprising a compound of formula (I):
R ¹ -L ¹ -R ² -L ² -R ³ (I)
(Where
R ¹ is interrupted by one or more of —O—, —S—, —C (O) —, —N (R ⁴ ) — or —C (O) N (R ⁴ ) —; and hydroxy, thiol A linear or branched C ₁ -C ₁₀ alkyl, alkenyl, or alkynyl chain substituted by two or more groups selected from amino, nitrogen oxides, phosphines or phosphine oxides;
L ¹ is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O)-, -C (O) N (R ⁴ ) -O- or-(CR ⁵ R ⁶ ) _n- ;
R ² is-[(CR ⁵ R ⁶ ) _n -X- (CR ⁵ R ⁶ ) _n ] _m- , X is O, S, C (= O) or N (R ⁴ ); An integer in the range 0-20;
L ² is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O)-, -C (O) N (R ⁴ ) -O- or-(CR ⁵ R ⁶ ) _n- ;
R ³ is — (CR ⁵ R ⁶ ) pR ⁷ , R ⁷ is —COOH, —OP (O) (OH) OH, amino, alkylamino, dialkylamino or trialkylamino; p is 0 1, 2, 3, 4, 5, or 6;
R ⁴ is H or C ₁ -C ₆ alkyl;
Each R ⁵ and each R ⁶ is independently selected from H, hydroxy, amino, thio, nitro, alkylamino, dialkylamino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl and heteroaryl;
Each n is independently 0, 1, 2, 3, 4, 5, or 6. ). A method to image a single particle:
Binding an affinity tag to a cell surface protein;
Contacting the cell with a composition comprising a valence-controlled semiconductor nanocrystal bound to exactly one compound having exactly one binding site for the affinity tag; and Imaging said semiconductor nanocrystals substantially simultaneously.   12. The method according to claim 11, wherein the affinity tag is biotin.   13. The method of claim 12, wherein binding the affinity tag to the cell surface protein comprises contacting a fusion protein comprising a receptor peptide (AP) sequence with a biotin ligase.   14. The method of claim 13, wherein the semiconductor nanocrystal is bound to exactly one monovalent avidin or exactly one monovalent streptavidin. 15. The method of claim 14, wherein the semiconductor nanocrystal comprises an outer layer comprising a compound of formula (I):
R ¹ -L ¹ -R ² -L ² -R ³ (I)
(Where
R ¹ is interrupted by one or more of —O—, —S—, —C (O) —, —N (R ⁴ ) — or —C (O) N (R ⁴ ) —; and hydroxy, thiol A linear or branched C ₁ -C ₁₀ alkyl, alkenyl, or alkynyl chain substituted by two or more groups selected from amino, nitrogen oxides, phosphines or phosphine oxides;
L ¹ is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O)-, -C (O) N (R ⁴ ) -O- or-(CR ⁵ R ⁶ ) _n- ;
R ² is-[(CR ⁵ R ⁶ ) _n -X- (CR ⁵ R ⁶ ) _n ] _m- , X is O, S, C (= O) or N (R ⁴ ); An integer in the range 0-20;
L ² is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O)-, -C (O) N (R ⁴ ) -O- or-(CR ⁵ R ⁶ ) _n- ;
R ³ is — (CR ⁵ R ⁶ ) pR ⁷ , R ⁷ is —COOH, —OP (O) (OH) OH, amino, alkylamino, dialkylamino or trialkylamino; p is 0 1, 2, 3, 4, 5, or 6;
R ⁴ is H or C ₁ -C ₆ alkyl;
Each R ⁵ and each R ⁶ is independently selected from H, hydroxy, amino, thio, nitro, alkylamino, dialkylamino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl and heteroaryl;
Each n is independently 0, 1, 2, 3, 4, 5, or 6. ). The compound of formula (I) is represented by the following formula:

Or the following formula:

16. The method of claim 15, comprising: A semiconductor nanocrystal comprising an outer layer comprising a compound of formula (I):
R ¹ -L ¹ -R ² -L ² -R ³ (I)
(Where
R ¹ is interrupted by one or more of —O—, —S—, —C (O) —, —N (R ⁴ ) — or —C (O) N (R ⁴ ) —; and hydroxy, thiol A linear or branched C ₁ -C ₁₀ alkyl, alkenyl, or alkynyl chain substituted by two or more groups selected from amino, nitrogen oxides, phosphines or phosphine oxides;
L ¹ is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O)-, -C (O) N (R ⁴ ) -O- or-(CR ⁵ R ⁶ ) _n- ;
R ² is-[(CR ⁵ R ⁶ ) _n -X- (CR ⁵ R ⁶ ) _n ] _m- , X is O, S, C (= O) or N (R ⁴ ); An integer in the range 0-20;
L ² is -C (O)-, -N (R ⁴ ) C (O)-, -C (O) N (R ⁴ )-, -O-, -N (R ⁴ )-, -ON ( R ⁴ ) C (O)-, -C (O) N (R ⁴ ) -O- or-(CR ⁵ R ⁶ ) _n- ;
R ³ is — (CR ⁵ R ⁶ ) pR ⁷ , R ⁷ is —COOH, —OP (O) (OH) OH, amino, alkylamino, dialkylamino or trialkylamino; p is 0 1, 2, 3, 4, 5, or 6;
R ⁴ is H or C ₁ -C ₆ alkyl;
Each R ⁵ and each R ⁶ is independently selected from H, hydroxy, amino, thio, nitro, alkylamino, dialkylamino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl and heteroaryl;
Each n is independently 0, 1, 2, 3, 4, 5, or 6. ). 18. The semiconductor nanocrystal according to claim 17, wherein R ¹ is HS—CH ₂ CH ₂ CH (SH) — (CH ₂ ) ₄ —. R ² is a poly (alkylene oxide), a semiconductor nanocrystal according to claim 17. R ² is a poly (ethylene glycol), a semiconductor nanocrystal according to claim 19, wherein. R ² has the formula _{- [CH 2 -O-CH 2} ] m - have, m is about 8, the semiconductor nanocrystal according to claim 17. 18. The semiconductor nanocrystal of claim 17, wherein R ³ is —CH ₂ —R ⁷ and R ⁷ is amino, alkylamino, dialkylamino or trialkylamino. R ⁷ is -COOH, semiconductor nanocrystal according to claim 17. R ³ is -CH ₂ COOH, semiconductor nanocrystal according to claim 17. R ¹ is _{HS-CH 2 CH 2 CH (} SH) - (CH 2) 4 - a and, R ² is poly (alkylene oxide), R ⁷ is -COOH, amino, alkylamino, dialkylamino or trialkyl 18. The semiconductor nanocrystal according to claim 17, which is amino. The compound has the following formula:

Or the following formula:

18. The semiconductor nanocrystal according to claim 17, comprising:

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